Polynucleotide construct capable of displaying fab in a cell-free translation system, and method for manufacturing and screening fab using same

ABSTRACT

The polynucleotide construct of (1) or (2) below is used to perform ribosome display, CIS display and/or mRNA display in order to screen a Fab against an antigen of interest: (1) a polynucleotide construct which monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself; and (2) a polynucleotide construct which comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, the first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of co-pending U.S. patentapplication Ser. No. 13/990,654, filed on Jul. 11, 2013, which is theU.S. national phase of International Patent Application No.PCT/JP2011/077725, filed Nov. 30, 2011, which claims the benefit ofJapanese Patent Application No. 2010-268763, filed on Dec. 1, 2010,which are incorporated by reference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 177,375 bytes ASCII (Text) file named“730386SequenceListing.txt,” created Aug. 18, 2017.

TECHNICAL FIELD

The present invention relates to a polynucleotide construct that candisplay Fab in a cell-free translation system, a kit comprising thesame, and a production method and a screening method for Fab using thesame.

BACKGROUND ART

Antibodies are glycoproteins produced by B cells, and have the functionto recognize molecules (antigens) such as proteins and bind thereto.Antibodies are produced in response to various internal and externalstimuli (antigens), and cooperate with immunocompetent cells toeliminate bacteria and viruses that invaded the body, thereby playing animportant role in the biological defense mechanism of vertebrates. Asingle type of B cell can produce only a single type of antibody, and asingle type of antibody can recognize only a single type of antigen. Inthe human body, several millions to several hundred millions of types ofB cells produce different antibodies to cope with numerous kinds ofantigens. These are collectively called immunoglobulin, which is one ofthe most abundant proteins in the blood and constitutes 20% by weight ofthe total plasma proteins. Antibodies are utilized, based on theirantigen specificities, as molecular-targeted agents and diagnosticagents.

A naturally-occurring antibody molecule forms a Y-shaped basic structureby association of two each of two types of polypeptide chains, the Lchain (light chain) and the H chain (heavy chain). The lower half of theY shape is composed of the Fc region, and the upper half of the Y shape,that is, the V-shaped portion, is composed of two identical Fab regions.The distal half of Fab is called the variable region (V region), andshows diversity in its amino acid sequence so that the antibodies canbind to various antigens. The variable regions in the L chain and the Hchain are called VL and VH, respectively. On the other hand, the Fc-sidehalf of Fab is called the constant region (C region), and shows lessvariability in its amino acid sequence. The constant regions in the Lchain and the H chain are called CL and CH1, respectively. Each regionin VL and VH that directly contacts with an antigen has especially highdiversity in its amino acid sequence, and called thecomplementarity-determining region (CDR). The other part is called theframework region (FR). Each of VL and VH has 3 CDRs (CDR1 to CDR3), and4 FRs (FR1 to FR4) surrounding these. The sequence diversity of CDR1 andCDR2 in naive B cells that have not been stimulated with an antigen isderived from the genomic DNA sequence (germline sequence). On the otherhand, sequences of CDR3 are newly formed by recombination reaction ofthe genomic DNA that occurs in the process of differentiation of Bcells, and hence its diversity is larger than those of the other CDRs.Moreover, in contrast to L-chain CDR3 famed by single recombinationreaction (V-J recombination), H-chain CDR3 is formed through two timesof recombination reaction (V-D recombination and D-J recombination), sothat the H chain has a larger diversity in the same CDR3. The 6 CDRsform a single continuous surface involved in antigen binding in thedistal part of Fab. In naturally-occurring antibodies, the functionaland structural unit involved in antigen binding is Fab. While each ofVL, VH, CL and CH1 constituting Fab forms an independent domain in termsof the spatial structure, they achieve high stability due tointeractions among the 4 domains. Although the Fc region is not directlyinvolved in binding of the antibody to an antigen, it is involved invarious effector functions (e.g., the antibody-dependent cell-mediatedcytotoxicity function).

Attempts are being made to prepare a gene library that expressesantibodies, in order to screen a gene encoding an antibody thatspecifically binds to a target antigen. In such a method, in order toobtain an antibody that specifically binds to a target antigen, anantibody library is expressed from DNA encoding the antibody library,and the expressed antibody library is brought into contact with thetarget antigen to select antibodies that specifically bind to the targetantigen, followed by amplification of the DNAs encoding the selectedantibodies. This cycle is repeated for screening antibodies. Since eachantibody selected by such a screening method using a display technologyis accompanied by information on the gene encoding its amino acidsequence, the selected antibody can be immediately prepared in a largeamount by genetic engineering based on the genetic information encodingthe antibody. Further, the amino acid sequence can also be easilydetermined by analysis of the genetic information.

An example of the antibody screening method using a display technologyis phage display reported in 1985 by G. Smith et al. (Non-patentDocument 1). Phage display is a technique in which a foreign protein isexpressed as a fusion protein with mainly a coat protein of afilamentous phage, and the polynucleotide encoding the foreign proteinof interest is selected. This method is widely used for, for example,selection of an antibody that specifically binds to an antigen molecule(Non-patent Documents 2 and 3). However, construction of a phage libraryrequires the step of transformation of E. coli, and this step limits thesize of the library. That is, due to the limit of efficiency oftransformation of E. coli, construction of a library having a diversityof, for example, more than 10¹⁰ requires several ten times to severalthousand times of transformation of E. coli by electroporation.Therefore, construction of a library larger than this is consideredunrealistic. Further, since translation into a protein is dependent onE. coli, the efficiency of expression of a protein harmful to E. coli asa host is remarkably low.

On the other hand, cell-free display systems represented by ribosomedisplay are techniques wherein proteins synthesized by a cell-freetranslation system are associated with polynucleotides encoding theproteins. Screening of an antibody using this system has been reported(Patent Document 1). However, what was actually prepared in this reportis a single-chain antibody (scFv) wherein the heavy-chain variableregion (VH) is linked to the light-chain variable region (VL) through alinker peptide, and no specific method for preparing Fab is disclosed.That is, it has been thought that Fab, which is constituted by twopeptide chains and has a larger molecular weight than scFv, is difficultto be handled in ribosome display because, for example, ribosome displayis generally a technique wherein a single molecule of RNA is coordinatedwith a single molecule of protein utilizing the 3′-end of the RNA, andsynthesis of a full-length peptide chain becomes more difficult as themolecular weight of the protein to be synthesized increases (Non-patentDocument 4). Further, since Fab is double-chained, the screeningefficiency might decrease due to occurrence of not only cis-associationof the H chain and the L chain displayed on the same RNA, but alsotrans-association of the H chain and the L chain on different RNAs.

As an attempt to use Fab in a cell-free display system, Yanagawa et al.reported a method wherein the H chain and the L chain constituting Fabare bicistronically expressed in a water-in-oil emulsion, and the Fab isassociated with the DNA encoding it utilizing the interaction betweenstreptavidin and biotin (Non-patent Document 4). However, since thismethod employs a water-in-oil emulsion, there are still problems in, forexample, that the size of the library which can be handled is limited;that, since Fab synthesized in a compartment is once dissociated fromDNA and mRNA and only one molecule of Fab is linked to the DNA, theremaining numerous Fab molecules cause competition; that the enrichmentratio is about 100, which is not very high; and that operations in theexperiment require high skill.

Further, there is a known method called look-through mutagenesis forincreasing the affinity of a target-substance-binding protein to itstarget substance, in which a library is prepared by introduction of asingle amino acid substitution into a target-substance-binding site in atarget-substance-binding protein, and the library is used to performfirst screening, followed by combining the obtained single amino acidsubstitutions and performing second screening to screen mutant proteinshaving improved affinity to the target substance (Patent Document 2).However, in this method, the sequences obtained by the first screeningare cloned, and mutant proteins are expressed from the obtained clonesto confirm their affinity to the target substance, followed byperforming the second screening. This is very laborious.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] JP 2008-271903 A-   [Patent Document 2] JP 2011-182794 A

Non-Patent Documents

-   [Non-patent Document 1] G. P. Smith et al. (1985) Science, vol.    228, p. 1315-1317-   [Non-patent Document 2] J. McCafferty et al., Nature (1990) vol.    348, p. 552-554-   [Non-patent Document 3] M. Hust et al., BMC Biotechnology, 2007, 7:    14-   [Non-patent Document 4] T. Sumida et al., Nucleic Acid Research,    2009, 37: 22, 147

SUMMARY OF THE INVENTION

Although screening of antibodies using the display technology hasalready been carried out as described above, it has been considered thatefficient screening of Fab having a large molecular weight and composedof two peptide chains is difficult with a cell-free display system suchas ribosome display. In view of this, the present invention aims toprovide a method wherein Fab is efficiently expressed in a cell-freetranslation system such as ribosome display and coordinated with thepolynucleotide encoding it in order to perform screening, and to providea polynucleotide construct therefor.

The present inventors intensively studied to solve the above problems.As a result, the present inventors discovered that Fab can beefficiently screened in a cell-free translation system by preparing apolynucleotide construct for bicistronic expression of the Fab H chainand the Fab L chain associated with their respective nucleotide sequenceinformation or a polynucleotide construct for monocistronic expressionof the Fab H chain and the Fab L chain associated with their nucleotidesequence information, and performing ribosome display and/or CIS displayusing it; and further discovered the Ymacs method, wherein the affinityof an antibody is remarkably improved by using this cell-free Fabdisplay system, thereby completed the present invention.

That is, the present invention provides the followings:

(Polynucleotide Construct to be Used for Cell-Free Display Method ofFab)

[1] A polynucleotide construct comprising a Fab first chain-codingsequence and a Fab second chain-coding sequence, wherein saidpolynucleotide construct expresses a Fab encoded by itself withoutdissociation, and maintains a complex with the Fab, when it isintroduced into a cell-free translation system containing ribosomes.

(Polynucleotide Construct (Monocistronic))

[2] The polynucleotide construct according to [1], wherein saidpolynucleotide construct monocistronically comprises a ribosome-bindingsequence, Fab first chain-coding sequence, linker peptide-codingsequence, Fab second chain-coding sequence and scaffold-coding sequencein this order, and further comprises at its 3′-end a structure necessaryfor maintaining a complex with the Fab encoded by itself.

(Polynucleotide Construct (Bicistronic))

[3] The polynucleotide construct according to [1], wherein saidpolynucleotide construct comprises a Fab first chain-expressing cistronand a Fab second chain-expressing cistron each containing aribosome-binding sequence, a Fab first chain-coding sequence or Fabsecond chain-coding sequence, and a scaffold-coding sequence in thisorder, said first Fab-expressing cistron further comprising at its3′-end a ribosome stall sequence, said Fab second chain-expressingcistron further comprising at its 3′-end a structure necessary formaintaining a complex with the Fab encoded by itself.

(Polynucleotide Construct to be Used for Ribosome/mRNA/CIS DisplayMethod)

[4] The polynucleotide construct according to [2] or [3], wherein saidstructure necessary for maintaining a complex with the Fab encoded byitself is a ribosome stall sequence, puromycin or a derivative thereof,or a DNA-binding protein-coding sequence and a binding sequence for saidDNA-binding protein.

(Polynucleotide Construct to be Used for Ribosome Display Method)

[5] The polynucleotide construct according to [4], wherein saidstructure necessary for maintaining a complex with the Fab encoded byitself is a ribosome stall sequence.[6] The polynucleotide construct according to [5], wherein said ribosomestall sequence is a SecM sequence, diproline sequence, or both of them.[7] The polynucleotide construct according to [5] or [6], wherein saidribosome stall sequence is composed of 2 to 4 repeats of the SecMsequence.[8] The polynucleotide construct according to any one of [4] to [7],wherein a stop codon is present in the 3′-side of said ribosome stallsequence.(Polynucleotide Construct to be Used for mRNA Display Method)[9] The polynucleotide construct according to [4], wherein saidstructure necessary for maintaining a complex with the Fab encoded byitself is puromycin or a derivative thereof.

(Polynucleotide Construct to be Used for CIS Display Method)

[10] The polynucleotide construct according to [4], wherein saidstructure necessary for maintaining a complex with the Fab encoded byitself is a DNA-binding protein-coding sequence and a binding sequencefor said DNA-binding protein.[11] The polynucleotide construct according to [10], wherein saidDNA-binding protein is a cis-binding protein which is never dissociatedduring transcription/translation reaction from the DNA molecule used asa template for said transcription/translation and which binds to saidbinding sequence for the DNA-binding protein located in the same DNAmolecule.[12] The polynucleotide construct according to [10], wherein saidDNA-binding protein is RepA encoded by Escherichia coli R1 plasmid andsaid binding sequence for the DNA-binding protein is a CIS-ori sequencelocated downstream of the RepA-coding sequence in the samepolynucleotide.[13] The polynucleotide construct according to any one of [1] to [12],wherein said polynucleotide construct is a library wherein said Fabfirst chain-coding sequence and said Fab second chain-coding sequencecomprise random sequences.[14] The polynucleotide construct according to [13], wherein saidlibrary comprising random sequences is a (i) naive library or (ii)focused library.[15] The polynucleotide construct according to [13] or [14], whereinsaid library comprising random sequences is a library comprising singleamino acid substitutions in the complementarity-determining region(s)(CDR(s)) of the Fab first chain and/or the Fab second chain.[16] A method for screening a Fab, said method comprising the steps of:

(i) introducing the polynucleotide construct according to any one of [1]to [15] into a cell-free translation system to synthesize Fabs, anddisplaying said synthesized Fabs on the polynucleotides encoding saidFabs;

(ii) bringing said Fabs displayed on said polynucleotides into contactwith an antigen;

(iii) selecting a Fab of interest that reacts with said antigen; and

(iv) amplifying the polynucleotide encoding said Fab of interest.

[17] The method for screening a Fab according to [16], said methodcomprising the steps of:

(I) providing a plurality of types of the polynucleotide constructaccording to [15], in each of which the Fab first chain-coding sequenceor the Fab second chain-coding sequence encodes an amino acid sequencecomprising a single amino acid substitution at a single position in aCDR in the amino acid sequence of the Fab first chain or the Fab secondchain of the parent antibody, such that single amino acid substitutionsare contained for a plurality of positions in the CDRs of the Fab firstchain and the Fab second chain;

(II) carrying out first screening wherein said steps (i) to (iv) arerepeated using said plurality of types of the polynucleotide construct,to screen a plurality of high-affinity Fabs;

(III) analyzing single amino acid substitutions at respective positionsin the CDRs of the Fab first chain and the Fab second chain in saidplurality of Fabs selected in said first screening step;

(IV) providing the polynucleotide construct according to [15] whereinthe Fab first chain-coding sequence and the Fab second chain-codingsequence encode amino acid sequences comprising combinations of thesingle amino acid substitutions identified in said first screening atsaid respective positions in the CDRs of the Fab first chain and Fabsecond chain sequences of the parent antibody; and

(V) carrying out second screening wherein said steps (i) to (iv) arerepeated using said polynucleotide construct, to screen a high-affinityFab.

[18] The method according to [16] or [17], wherein said in vitrotranslation system is composed of factors independently purified.[19] The method according to [18], wherein said in vitro translationsystem contains at least one component selected from the groupconsisting of initiation factors, elongation factors, aminoacyl-tRNAsynthetase and methionyl-tRNA transformylase.[20] The method according to [18] or [19] wherein said in vitrotranslation system does not contain a releasing factor.[21] The method according to [16] or [17], wherein said in vitrotranslation system is a cell extract containing ribosomes.[22] A method for producing a Fab, said method comprising the step ofintroducing the polynucleotide construct according to any one of [1] to[15] into an in vitro translation system to produce a Fab.[23] A kit for producing or screening a Fab, said kit comprising thepolynucleotide construct according to any one of [1] to [15].[24] A method for maximizing the affinity of a target-substance-bindingprotein to a target substance, said method comprising the steps of:

(I) constructing single-position libraries wherein one amino acid amongall amino acid positions constituting a target-substance-binding site ina target-substance-binding protein is randomized to all the 20 types ofnaturally-occurring amino acids, to provide as many single-positionlibraries as the number of the all amino acid positions;

(II) constructing a primary library by integrating all of, or anappropriate unit of, these single-position libraries;

(III) selecting said primary library using a protein display systembased on the affinity to a target;

(IV) determining the polynucleotide sequence information of saidselected sample of the primary library;

(V) extracting single amino acid substitutions frequently observed insaid nucleotide sequence information;

(VI) constructing a secondary library comprising combinations of saidfrequently observed single amino acid substitutions; and

(VII) selecting said secondary library using a protein display systembased on the affinity to a target.

[25] The method according to [24], wherein the step of determining thepolynucleotide sequence information of said selected sample of theprimary library is carried out using a next-generation sequencer.[26] The method according to [24] or [25], wherein saidtarget-substance-binding protein is a full-length antibody or anantibody fragment and said target-substance-binding site is a CDRregion.[27] The method according to any one of [24] to [26], wherein saidprotein display system is ribosome display, CIS display, mRNA display,phage display, bacterial surface display, yeast cell surface display, orcell surface display with a higher eukaryote.

According to the present invention, a Fab of interest can be screened byefficiently expressing Fab without dissociation from the polynucleotidein a cell-free display system. Since the method of the present inventioncan be carried out in a cell-free translation system, the operation issimple, and the screening can be carried out in a short period. Alarge-scale library with a level of not less than 10¹² can be easilyconstructed, and, by this, highly efficient screening is possible.Further, according to the Ymacs method of the present invention, a largenumber of Fabs having several hundred to not less than a thousand timeshigher affinity to an antigen can be obtained as compared to error-pronePCR, CDR shuffling and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, the symbols on mRNA and DNA represent the followings:

“VL”, coding sequence in the L chain variable region of Fab;

“CL”, coding sequence in the L chain constant region of Fab;

“VH”, coding sequence in the H chain variable region of Fab;

“CH1”, coding sequence in the H chain constant region of Fab;

“Linker”, coding sequence of the linker peptide;

“RBS (o)”, ribosome-binding site;

“Pu”, puromycin or a derivative thereof;

“Pro Δ”, promoter;

“RNAP”, RNA polymerase;

“RSS(S)”, ribosome stall sequence; and

“LP(P)”, leader peptide sequence for secretory expression.

FIG. 1 is a schematic diagram showing display of Fab on a monocistronicpolynucleotide construct (an embodiment utilizing ribosome display) (RBS(o) represents the ribosome-binding site; the same applies hereinafter).

FIG. 2 is schematic diagram showing display of Fab on a monocistronicpolynucleotide construct (an embodiment utilizing mRNA display).

FIG. 3 is schematic diagram showing display of Fab on a monocistronicpolynucleotide construct (an embodiment utilizing CIS display) (Δrepresents a promoter; the same applies hereinafter).

FIG. 4 is a schematic diagram showing display of Fab on a bicistronicpolynucleotide construct (an embodiment utilizing ribosome display forboth the 5′-side cistron and the 3′-side cistron).

FIG. 5 is a schematic diagram showing display of Fab on a bicistronicpolynucleotide construct (an embodiment utilizing ribosome display forthe 5′-side cistron and utilizing mRNA display for the 3′-side cistron).

FIG. 6 is a schematic diagram showing display of Fab on a bicistronicpolynucleotide construct (an embodiment utilizing ribosome display forthe 5′-side cistron and utilizing CIS display for the 3′-side cistron).

FIG. 7 is a schematic diagram showing a method for screening (ribosomedisplay) of Fab.

FIG. 8 is a schematic diagram showing a method for screening (CISdisplay) of Fab.

FIG. 9 is a schematic diagram showing a pTrc-Fab bicistronic Fabsecretory expression unit.

FIG. 10 is a photograph showing staining of model Fabs with CBB. The twolanes in the left side show markers.

FIG. 11 is a graph showing the result of ELISA (low concentration, Her2antigen) of model Fabs.

FIG. 12 is a graph showing the result of ELISA (high concentration) ofmodel Fabs.

FIG. 13 is a schematic diagram showing a DNA fragment for bicistronicFab-PRD (polynucleotide construct). RSS represents a ribosome stallsequence (the same applies to FIG. 14).

FIG. 14 is a schematic diagram showing a DNA fragment for monocistronicFab-PRD (polynucleotide construct).

FIG. 15 is a graph showing the result of evaluation of biotinylatedantigens by sandwich ELISA. BSA represents bovine serum albumin(control), and stAv represents streptavidin.

FIG. 16 is an electrophoretogram showing the result of Fab-HHxhoenrichment in bicistronic Fab-PRD (for only the middle portion). WBrepresents a washing buffer.

FIG. 17 is an electrophoretogram showing the result of Fab-HHxhoenrichment in bicistronic Fab-PRD (for the full-length sequence).

FIG. 18 is an electrophoretogram showing the result of Fab-HHxhoenrichment in monocistronic Fab-PRD (for the full-length sequence).

FIG. 19 is an electrophoretogram (Western blot) showing the result ofdetection of in vitro translation products of monocistronic Fab-PRD.

FIG. 20 is a diagram showing the relationship between the copy number ofSecM and the recovery in monocistronic Fab-PRD.

FIG. 21 is a diagram showing the relationship between the length of thelinker and the enrichment ratio in monocistronic Fab-PRD.

FIG. 22 is an electrophoretogram showing recovery of Fab-TT from apopulation of 400 molecules of Fab-TT and 1×10¹² molecules of Fab-HH inmonocistronic Fab-PRD.

FIG. 23 is a diagram showing a summary of synthesis of asingle-position/single-amino acid substitution library.

FIG. 24 is a diagram showing sequences of the CDRs in a Ymacs-primarylibrary. NNK in the tables indicates the position into which a singleamino acid substitution is to be introduced.

FIG. 25 is a summary of the mutations selected in the first screening.PA in the Table indicates the parent amino acid.

FIG. 26 is a diagram showing the mutations employed in theYmacs-secondary library, and codons corresponding thereto.

FIG. 27 is a diagram showing the method for constructing theYmacs-secondary library.

FIG. 28 is a schematic diagram showing conversion of a monocistronicsecretory Fab expression vector to a bicistronic Fab secretoryexpression vector.

FIG. 29 is a diagram showing the sequences of the CDRs of the Fabsselected by screening of the Ymacs-secondary library.

FIG. 30 is a diagram showing the result of koff measurement by SPRanalysis of Fabs selected by screening of the Ymacs-secondary library.

FIG. 31 is a diagram showing the result of KD measurement by SPRanalysis of Fab-TT as a parent antibody and its affinity-improved mutantYmacs #10.

FIG. 32 is a diagram showing the result of KD measurement by KinExAanalysis of the affinity-improved mutants Ymacs #10 and Ymacs #19.

FIG. 33 is an electrophoretogram showing the result of Fab-HHxhoenrichment (for full length) in CIS display.

FIG. 34 is an electrophoretogram showing the result of study on theeffect of the reaction time of transcription/translation in Fab-HHxhoenrichment (for full length) in CIS display.

EMBODIMENTS FOR CARRYING OUT THE INVENTION <Polynucleotide Construct>

The polynucleotide construct of the present invention comprises a Fabfirst chain-coding sequence and a Fab second chain-coding sequence,wherein the polynucleotide construct expresses the Fab encoded by itselfwithout dissociation, and maintains a complex with a Fab, when it isintroduced into a cell-free translation system containing ribosomes. TheFab first chain and the Fab second chain herein mean the two chainsconstituting Fab, and, usually, one of these is the Fab H chain and theother is the Fab L chain. However, each chain may be a chimeric chain ofthe H chain and the L chain. The Fab H chain means a protein containingthe H-chain variable region (VH) and the H-chain constant region 1(CH1), and the Fab L chain means a protein containing the L-chainvariable region (VL) and the L-chain constant region (CL).

The team “maintains a complex with the Fab” means that the Fab isexpressed in a state where the Fab is linked to the polynucleotideconstruct, and the complex is maintained. This is also expressed as “theFab is displayed on the polynucleotide”.

Polynucleotide Construct (Monocistronic)

The polynucleotide construct of the first embodiment of the presentinvention monocistronically comprises a ribosome-binding sequence, Fabfirst chain-coding sequence, linker peptide sequence, Fab secondchain-coding sequence and scaffold-coding sequence in this order. Thepolynucleotide construct further comprises a structure necessary formaintaining a complex with the Fab encoded by itself, at the 3′-end ofthe coding region (cistron).

The ribosome-binding sequence means the sequence upstream of theinitiation codon, where a ribosome is bound to initiate translation. Incases where a ribosome derived from E. coli is used as a cell-freetranslation system, the Shine-Dalgarno (SD) sequence is preferably usedas the ribosome-binding sequence. As the SD sequence, AGGAGGT isgenerally known, but a modified sequence may be used as long as aribosome can bind thereto. The ribosome-binding sequence may beappropriately selected depending on the host, and is not limited to theSD sequence.

Since sequences of the common region in Fab have been disclosed (Sakanoet al., Nature (1980) vol. 286, p. 676; Ellison et al., Nucleic AcidsRes. (1982) vol. 10, p. 4071; Huck et al., Nucleic Acids Res. (1986)vol. 14, p. 1779; Hieter et al., J. Biol. Chem. (1982) vol. 257, p.1516; and Max et al., Cell (1980) vol. 22, p. 197), a Fab chain-codingsequence can be obtained by designing primers based on these sequencesand amplifying the Fab H chain-coding sequence and the Fab Lchain-coding sequence. Primers for cloning of the variable region andthe constant region of the antibody are known (Marks et al., J. Mol.Biol. (1991) vol. 222, p. 581; Welschof et al., J. Immunol. Methods(1995) vol. 179, p. 203; Campbell et al., Mol. Immunol. (1992) vol. 29,p. 193). Alternatively, a Fab chain-coding sequence can be artificiallysynthesized in consideration of the codon bias and RNA processing of thehost to be used in large-scale expression.

In cases where a specific Fab is to be expressed, the sequence may beamplified from a template of interest. Further, in cases where a Fabagainst an antigen of interest is to be screened, a library containingrandom Fab chain-coding sequences may be used. In cases where a Fab thatbinds to an antigen of interest is to be newly obtained, a naive librarymay be used. Further, in cases where a specific Fab which has alreadybeen obtained is to be optimized for a certain purpose, a focusedlibrary may be used.

In order to provide a very wide range of search space for newlyobtaining a Fab that binds to an antigen of interest, various forms ofnaive libraries can be constructed as exemplified below. For example, itis possible to construct a natural naive library utilizing the antibodydiversity produced by natural B cells, by performing reversetranscription-PCR using mRNA collected from B cells as a startingmaterial to amplify cDNA fragments of the VH region and/or the VL regionconstituting Fab (Clackson et al., Nature (1991) vol. 352, p. 624-628).It is also possible to construct a semisynthetic naive library byartificially synthesizing the framework region in the VH region and/orthe VL region and incorporating various naturally-occurring CDRsequences into the CDR regions (Soderlind et al., Nat. Biotechnol.(2000) vol. 18, p. 852-856). It is also possible to construct asemisynthetic naive library by incorporating a naturally-occurringsequence into the L chain (VL-CL), an artificial sequence into CDRs 1-2in the H chain, and a naturally-occurring sequence into CDR3 in the Hchain, of Fab (Hoet et al., Nat. Biotechnol. (2005) vol. 23, p.344-348). It is also possible to construct a completely artificial naivelibrary by artificially synthesizing the full region of the codingsequence, while giving diversity to the CDRs (Knappik et al., J. Mol.Biol. (2000) vol. 296, p. 57-86). In cases where the CDR sequences areartificially synthesized, the frequencies of amino acids that appear ateach amino acid position can be controlled depending on the lengths ofCDRs based on the result of analysis of naturally-occurring CDRsequences.

In cases of a focused library, various forms of the library can beconstructed based on the sequence of a specific parent Fab which hasalready been obtained, such that the library contains, as a majorcomponent, mutants that are similar to, but different from, the parentFab sequence. A focused library may be used for optimization for thepurposes of improving the affinity, optimizing the specificity,humanizing an animal-derived antibody, eliminating a disadvantageousamino acid or sequence, improving the stability, improving the physicalproperties, and the like, while maintaining the antigen-binding propertyof the parent Fab.

For example, error-prone PCR may be used for randomly and sporadicallyintroducing one or several single amino acid substitutions over theentire VH region and/or VL region (Boder et al., Proc Natl Acad Sci USA(2000) vol. 97, p. 10701-10705). Error-prone PCR utilizes the phenomenonthat errors can be made to easily occur by controlling theconcentrations of the 4 types of deoxynucleotides as substrates and thetype and concentration of the divalent cation added. It is also possibleto use artificially-designed mutation-introducing primers for targetinga specific amino acid(s) constituting Fab to introduce one or severalsingle amino acid substitutions in consideration of the amino acidcomposition (Rajpal et al., Proc Natl Acad Sci USA (2005) vol. 102, p.8466-8471). Further, it is also possible to use artificially-designedmutation-introducing primers for randomizing the sequence(s) of about 1to 3 CDR(s) while the sequences of about 3 to 5 CDRs are maintainedamong the total of 6 CDRs, such that the randomization occurs not forsingle amino acids but for entire CDR sequences (Lee et al., Blood(2006) vol. 108, p. 3103-3111). Further, it is also possible to fix oneof the L chain and the H chain while the other is randomized utilizingvarious naturally-occurring sequences (Kang et al., Proc Natl Acad SciUSA (1991) vol. 88, p. 11120-11123).

As the polynucleotide construct of the present invention, a librarywherein one or more amino acids in the complementarity determiningregions (CDRs) of the Fab first chain and/or the Fab second chaincontain a single amino acid substitution(s) is preferred. Such a librarycan be prepared by, for example, PCR using primers designed such thatthe single amino acid substitution(s) is/are introduced.

By performing screening using such a polynucleotide construct library,an antibody against an antigen of interest can be obtained.

The sequence encoding the linker peptide that links the Fab firstchain-coding sequence and the Fab second chain-coding sequence ispreferably a sequence encoding a water-soluble polypeptide composed of15 to 120 amino acid residues, more preferably a sequence encoding awater-soluble polypeptide composed of 20 to 30 amino acids in view ofthe screening efficiency of the library. Examples of the sequenceinclude an amino acid sequence into which Arg was introduced forincreasing the water solubility, and the sequence may also be oneencoding the so-called GS linker, which mainly contains glycine andserine.

The sequence encoding the linker peptide may be placed between proteaserecognition sequences. By this, after translation of the polynucleotideconstruct into an amino acid sequence, the linker peptide is cleaved byprotease, resulting in the display of a natural Fab. Examples of theprotease recognition sequence include the enterokinase recognitionsequence DDDDK (SEQ ID NO:31) and the factor Xa recognition sequenceIEGR (SEQ ID NO:32).

Examples of the scaffold-coding sequence linked to the 3′-side of theFab chain-coding sequence include those encoding an amino acid sequencehaving a length sufficient as a scaffold with which the first chain andthe second chain of Fab can be translated and accurately folded on aribosome, DNA and/or mRNA to form a complex and react with an antigen.The scaffold-coding sequence is a sequence preferably encoding at least15 amino acids, more preferably encoding 15 to 120 amino acids. Thescaffold sequence encoded preferably has high water solubility and doesnot form a special spatial structure. Specific examples of such asequence which may be used include the so-called GS linker, which mainlycontains glycine and serine, and partial sequences of gene III in phages

As the structure necessary for maintaining a complex with the Fab chainencoded by itself, the construct may, for example, have a ribosome stallsequence at the 3′-end of the cistron (in cases of ribosome display);have a DNA-binding protein-coding sequence and a binding sequence forthe DNA-binding protein (in cases of CIS display); or have puromycin ora derivative thereof added to the 3′-end (in cases of mRNA display). Bythis, the expressed Fab chain forms a complex with the polynucleotide,and hence the nucleotide sequences of the first and Fab secondchain-coding sequences are physically associated with the amino acidsequences encoded thereby.

Polynucleotide Construct (Monocistronic) to be Subjected to RibosomeDisplay

Examples of the ribosome stall sequence include the sequence encodingSecM of E. coli. The SecM sequence is also called the SecM stallsequence, and is reported to cause translation arrest inside theribosome (FXXXXWIXXXXGIRAGP, SEQ ID NO:30). Since, by introduction ofthis sequence, the complex of mRNA, ribosome and fusion protein can beefficiently maintained (Nakatogawa et al., Mol. Cell (2006) vol. 22, p.545-552), the Fab chain can be associated with the nucleotide sequenceencoding it. Two or more SecM sequences may be linked, and the number ofSecM sequences to be linked is preferably 2 to 4, more preferably 2.

Further, a polyproline sequence such as diproline may be used as theribosome stall sequence, and such a sequence may be used either alone orin combination with a SecM sequence(s).

In the 3′-side of the ribosome stall sequence, a stop codon ispreferably placed in the same reading frame.

It is also possible to simply delete the stop codon instead of employinga ribosome stall sequence, for performing ribosome display.

The Fab first chain-coding sequence, linker-coding sequence, Fab secondchain-coding sequence, scaffold-coding sequence and ribosome stallsequence are linked together in the same reading frame. The term “linkedtogether in the same reading frame” herein means that these componentsare linked together such that they are translated as a fusion protein.The Fab first chain-coding sequence, linker-coding sequence, Fab secondchain-coding sequence, scaffold-coding sequence and ribosome stallsequence may be linked together either directly, or via a tagsequence(s) and/or an arbitrary polypeptide sequence(s) placed between,before and/or after the components.

An example of the polynucleotide construct of the first embodiment ofthe present invention wherein ribosome display is utilized is shown inFIG. 1.

SEQ ID NO:19 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 9 to 31); aribosome-binding sequence (SD sequence, nucleotide positions 81 to 87);and an anti-Her2 Fab L+H chain-expressing cistron (nucleotide positions94 to 2220, see SEQ ID NO:20 for its amino acid sequence) containing ananti-Her2 Fab L chain-coding sequence, FLAG tag, linker sequence (GSlinker), FLAG tag, anti-Her2 Fab H chain-coding sequence, His tag,scaffold sequence (GS linker) and ribosome stall sequence(secM+diproline).

SEQ ID NO:21 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 9 to 31); aribosome-binding sequence (SD sequence, nucleotide positions 81 to 87);and an anti-INFαR Fab L+H chain-expressing cistron (nucleotide positions94 to 2226, see SEQ ID NO:22 for its amino acid sequence) containing ananti-INFαR Fab L chain-coding sequence, FLAG tag, linker sequence (GSlinker), FLAG tag, anti-INFαR Fab H chain-coding sequence, His tag,scaffold sequence (GS linker) and ribosome stall sequence(secM+diproline).

However, needless to say, the polynucleotide construct of the presentinvention is not limited to these.

Polynucleotide Construct (Monocistronic) to be Subjected to mRNA Display

The amino acid sequence may be physically associated with the nucleotidesequence encoding it by utilizing, instead of the ribosome stallsequence, puromycin or a derivative thereof to allow formation of acomplex between the Fab and the polynucleotide (mRNA display). That is,puromycin or a derivative thereof is linked to the 3′-end of thepolynucleotide construct via a spacer, and the C-terminus of thetranslation product is covalently bound to the puromycin or a derivativethereof to allow formation of a complex between Fab and thepolynucleotide. By this, association of the amino acid sequence of theFab chain with the nucleotide sequence encoding it is possible.

As the puromycin or a derivative thereof, puromycin; and puromycinderivatives such as ribocytidyl puromycin, deoxycytidyl puromycin anddeoxyuridyl puromycin; are especially preferred.

Examples of the spacer to be used for linking the puromycin or aderivative thereof to the 3′-end of the polynucleotide construct includemacromolecular substances such as polyethylene and polyethylene glycol,and derivatives thereof; and biomacromolecular substances such asoligonucleotides and peptides, and derivatives thereof; as described inWO98/16636. Among these, polyethylene glycol is preferred.

In another embodiment of mRNA display, the amino acid sequence may bephysically associated with the nucleotide sequence encoding it bylinking a streptavidin-coding sequence to the 3′-end side of thepolynucleotide construct and further linking biotin to the 3′-end, toallow binding of the streptavidin portion of the translated protein tothe biotin, resulting in formation of a complex between Fab and thepolynucleotide.

An example of the polynucleotide construct of the first embodiment ofthe present invention wherein mRNA display is utilized is shown in FIG.2.

Polynucleotide Construct (Monocistronic) to be Subjected to CIS Display

Further, the amino acid sequence may be physically associated with thenucleotide sequence encoding it by utilizing a DNA-bindingprotein-coding sequence and the binding sequence for the DNA-bindingprotein instead of the ribosome stall sequence, to allow formation of acomplex between Fab and the polynucleotide (CIS display, WO2004/22746).More specifically, a DNA-binding protein-coding sequence and the bindingsequence for the DNA-binding protein are linked downstream of thescaffold sequence of the polynucleotide construct, and, Fab, thescaffold sequence and the DNA-binding protein are expressed as a fusionprotein. Since the DNA-binding protein binds to the binding sequence forthe DNA-binding protein downstream of the 3′-side cistron, a complexbetween Fab and the polynucleotide is formed, and hence the amino acidsequence of the Fab chain can be associated with the nucleotide sequenceencoding it. Examples of the DNA-binding protein herein include the RepAprotein, which has a cis-type binding mode wherein a DNA-binding proteinis bound to the binding sequence for the DNA-binding protein displayedon the same DNA molecule without dissociation, during thetranscription/translation reaction, from the DNA molecule used as thetemplate for the transcription/translation. Examples of the RepA-bindingsequence include the CIS sequence and the following ori sequence (Proc.Natl. Acad. Sci. U.S.A., vol. 101, p. 2806-2810, 2004; and JapaneseTranslated PCT Patent Application Laid-open No. 2005-537795). Otherexamples of the DNA-binding protein having a cis-type binding modeinclude the RecC protein encoded by E. coli Ti plasmid (Pinto, et al.,Mol. Microbiol. (2011) vol. 81, p. 1593-1606), A protein of φX174 phage(Francke, et al., Proc Natl Acad Sci USA (1972) vol. 69, p. 475-479) andQ protein of λ phage (Echols, et al., Genetics (1976) vol. 83, p. 5-10),each of which may be used in combination with its binding sequence. Incases where a cell-free translation system wherein transcription iswell-coupled with translation is employed, the synthesized protein isreleased in the vicinity of the transcription termination site, so thata DNA protein generally considered to have a trans-type binding mode,such as a DNA-binding domain of a nuclear receptor including theestrogen receptor, or a DNA-binding domain of LexA or Gal4 used for thetwo-hybrid system, may be used in combination with its binding sequence.

An example of the polynucleotide construct of the first embodiment ofthe present invention wherein CIS display is utilized is shown in FIG.3.

SEQ ID NO:70 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 612 to 639); aribosome-binding sequence (SD sequence, nucleotide positions 672 to675); an anti-Her2 Fab L+H chain-expressing cistron (nucleotidepositions 689 to 3322, see SEQ ID NO:71 for its amino acid sequence)containing an anti-Her2 Fab L chain-coding sequence, FLAG tag, linkersequence (GS linker), FLAG tag, anti-Her2 Fab H chain-coding sequence,His tag, scaffold sequence (GS linker) and RepA-coding sequence; andCIS-ori (nucleotide positions 3326 to 4100).

SEQ ID NO:72 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 612 to 639); aribosome-binding sequence (SD sequence, nucleotide positions 672 to675); an anti-INFαR Fab L+H chain-expressing cistron (nucleotidepositions 689 to 3328, see SEQ ID NO:73 for its amino acid sequence)containing an anti-INFαR Fab L chain-coding sequence, FLAG tag, linkersequence (GS linker), FLAG tag, anti-INFαR H chain-coding sequence, Histag, scaffold sequence (GS linker) and RepA-coding sequence; and CIS-ori(nucleotide positions 3332 to 4106).

However, needless to say, the polynucleotide construct of the presentinvention is not limited to these.

Polynucleotide Construct (Bicistronic)

The polynucleotide construct of the second embodiment of the presentinvention comprises a Fab first chain-expressing cistron and a Fabsecond chain-expressing cistron each containing a ribosome-bindingsequence, a Fab first chain-coding sequence or Fab second chain-codingsequence, and a scaffold-coding sequence in this order. The Fab firstchain-expressing cistron (Fab chain-expressing cistron in the 5′-side)further comprises at its 3′-end a ribosome stall sequence, and the Fabsecond chain-expressing cistron (Fab chain-expressing cistron in the3′-side) further comprises at its 3′-end side a structure necessary formaintaining a complex with the Fab encoded by itself. Examples of thestructure necessary for maintaining a complex with the Fab encoded byitself herein include a ribosome stall sequence; a DNA-bindingprotein-coding sequence and a binding sequence for the DNA-bindingprotein; and puromycin or a derivative thereof; as described above.

That is, the Fab first chain-expressing cistron comprises a Fab firstchain-coding sequence and a scaffold-coding sequence in the 3′-side of aribosome-binding sequence in this order, and the Fab secondchain-expressing cistron comprises a Fab second chain-coding sequenceand a scaffold-coding sequence in the 3′-side of a ribosome-bindingsequence in this order. The Fab first chain and the Fab second chain maybe either the H chain and the L chain, respectively, or the L chain andthe H chain, respectively, or each of the chains may be a chimeric chainbetween the H chain and the L chain.

The length between the Fab first chain-expressing cistron and the Fabsecond chain-expressing cistron is not restricted as long as an intervalcan be secured when translation has once terminated in the Fab firstchain-expressing cistron and the ribosome is stalled, so that theribosome can bind to the ribosome-binding sequence in the Fab secondchain-expressing cistron. The length is preferably 50 to 200 bp.

The ribosome-binding sequence and the Fab chain-coding sequence may bethose described above.

Examples of the scaffold-coding sequence linked to the 3′-side of eachFab chain-coding sequence include those encoding an amino acid sequencehaving a length sufficient as a scaffold with which the H chain or the Lchain of Fab can be translated and precisely folded to form a complex ona ribosome, DNA and/or mRNA, and react with an antigen. Thescaffold-coding sequence is a sequence preferably encoding at least 15amino acids, more preferably encoding 15 to 120 amino acids. Thescaffold sequence encoded preferably has high water solubility and doesnot form a special spatial structure. Specific examples of such asequence which may be used include the so-called GS linker, which mainlycontains glycine and serine, and partial sequences of gene III in phages

Polynucleotide Construct (Bicistronic) to be Subjected to RibosomeDisplay

In the polynucleotide construct of the second embodiment of the presentinvention, in order to allow formation of a complex between mRNA and apolypeptide (the first chain or the second chain of Fab) on a ribosome,a ribosome stall sequence as described above is placed at the 3′-end ofthe Fab first chain-expressing cistron. A stop codon is preferablyplaced in the 3′-side of the ribosome stall sequence in the same readingframe. It is also possible to simply delete the stop codon instead ofemploying a ribosome stall sequence, for performing ribosome display.

The Fab first chain-coding sequence or Fab second chain-coding sequence,scaffold-coding sequence and ribosome stall sequence are linked togetherin the same reading frame. The term “linked together in the same readingframe” herein means that these components are linked together such thatthey are translated as a fusion protein. The Fab first chain-codingsequence or Fab second chain-coding sequence, scaffold-coding sequenceand ribosome stall sequence may be linked together either directly, orvia a tag sequence(s) and/or an arbitrary polypeptide sequence(s) placedbetween, before and/or after the components.

An example of the polynucleotide construct of the second embodiment ofthe present invention wherein ribosome display is utilized for both theFab first chain-expressing cistron and the Fab second chain-expressingcistron is shown in FIG. 4.

SEQ ID NO:13 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 9 to 31); aribosome-binding sequence (SD sequence, nucleotide positions 81 to 87);an anti-Her2 Fab L chain-expressing cistron (nucleotide positions 94 to1158, see SEQ ID NO:14 for its amino acid sequence) containing ananti-Her2 Fab L chain-coding sequence, FLAG tag, scaffold sequence (GSlinker) and ribosome stall sequence (secM+diproline); a ribosome-bindingsequence (SD sequence, base positions 1191 to 1197); and an anti-Her2Fab H chain-expressing cistron (nucleotide positions 1264 to 2364, seeSEQ ID NO:15 for its amino acid sequence) containing an anti-Her2 Fab Hchain-coding sequence, His tag, scaffold sequence (GS linker) andribosome stall sequence (secM+diproline).

SEQ ID NO16 shows the nucleotide sequence of a polynucleotide constructcontaining: a promoter sequence (nucleotide positions 9 to 31); aribosome-binding sequence (SD sequence, nucleotide positions 81 to 87);an anti-TNFα receptor (TNFαR) Fab L chain-expressing cistron (nucleotidepositions 94 to 1158, see SEQ ID NO:17 for its amino acid sequence)containing an anti-TNFαR Fab L chain-coding sequence, FLAG tag, scaffoldsequence (GS linker) and ribosome stall sequence (secM+diproline); aribosome-binding sequence (SD sequence, base positions 1191 to 1197);and an anti-TNFαR Fab H chain-expressing cistron (nucleotide positions1264 to 2370, see SEQ ID NO:18 for its amino acid sequence) containingan anti-TNFαR Fab H chain-coding sequence, His tag, scaffold sequence(GS linker) and ribosome stall sequence (secM+diproline).

However, needless to say, the polynucleotide construct of the presentinvention is not limited to these.

Polynucleotide Construct (Bicistronic) to be Subjected to mRNA Display

In terms of the Fab second chain-expressing cistron (the Fabchain-expressing cistron in the 3′-side), puromycin or a derivativethereof may be utilized to allow formation of a complex between the Fabchain and the polynucleotide in order to physically associate the aminoacid sequence with the nucleotide sequence encoding it (mRNA display).That is, puromycin or a derivative thereof is linked to the end of the3′-side cistron, that is, to the 3′-end of the polynucleotide construct,via a spacer, and, when the 3′-side cistron is translated, theC-terminus of the translation product is covalently bound to thepuromycin or a derivative thereof to allow formation of a complexbetween the Fab chain and the polynucleotide. By this, association ofthe amino acid sequence of the Fab chain with the nucleotide sequenceencoding it is possible.

An example of the polynucleotide construct of the second embodiment ofthe present invention wherein ribosome display is utilized for the Fabfirst chain-expressing cistron and mRNA display is utilized for the Fabsecond chain-expressing cistron is shown in FIG. 5.

Polynucleotide Construct (Bicistronic) to be Subjected to CIS Display

In terms of the Fab second chain-expressing cistron (the Fabchain-expressing cistron in the 3′-side), a DNA-binding protein-codingsequence and the binding sequence for the DNA-binding protein may beutilized to allow formation of a complex between the Fab chain and thepolynucleotide in order to physically associate the amino acid sequencewith the nucleotide sequence encoding it (CIS display, WO2004/22746).More specifically, a DNA-binding protein-coding sequence and the bindingsequence for the DNA-binding protein are linked to the 3′-end-side ofthe Fab second chain-expressing cistron, that is, downstream of the Fabsecond chain-coding sequence and the scaffold sequence of the Fab secondchain-expressing cistron, and the Fab second chain-expressing cistron isexpressed as a fusion protein of the Fab second chain, scaffold sequenceand DNA-binding protein. The DNA-binding protein is bound to the bindingsequence for the DNA-binding protein located downstream of the Fabsecond chain-expressing cistron to allow formation of a complex betweenthe Fab second chain and the polynucleotide. By this, association of theamino acid sequence of the Fab second chain with the nucleotide sequenceencoding it is possible. Examples of the DNA-binding protein hereininclude the RepA protein, which has the so-called cis-type binding modewherein a DNA-binding protein is bound to the binding sequence for theDNA-binding protein present on the same DNA molecule withoutdissociation, during the transcription/translation reaction, from theDNA molecule used as the template for the transcription/translation.Examples of the RepA-binding sequence include the CIS sequence and thefollowing ori sequence (Proc. Natl. Acad. Sci. U.S.A., vol. 101, p.2806-2810, 2004; and Japanese Translated PCT Patent ApplicationLaid-open No. 2005-537795). Other examples of the DNA-binding proteinhaving a cis-type binding mode include the RecC protein encoded by E.coli Ti plasmid (Pinto, et al., Mol. Microbiol. (2011) vol. 81, p.1593-1606), A protein of φX174 phage (Francke, et al., Proc Natl AcadSci USA (1972) vol. 69, p. 475-479) and Q protein of λ phage (Echols, etal., Genetics (1976) vol. 83, p. 5-10), each of which may be used incombination with its binding sequence. In cases where a cell-freetranslation system wherein transcription is well-coupled withtranslation is employed, the synthesized protein is released in thevicinity of the transcription termination site, so that a DNA proteingenerally considered to have a trans-type binding mode, such as aDNA-binding domain of a nuclear receptor including the estrogenreceptor, or a DNA-binding domain of LexA or Gal4 used for thetwo-hybrid system, may be used in combination with its binding sequence.

An example of the polynucleotide construct of the second embodiment ofthe present invention wherein ribosome display is utilized for the Fabfirst chain-expressing cistron and CIS display is utilized for the Fabsecond chain-expressing cistron is shown in FIG. 6.

The polynucleotide construct of the present invention may be eithermRNA, or DNA from which mRNA is transcribed. In cases where thepolynucleotide construct is mRNA, the term “expresses Fab” meanstranslation of mRNA into the Fab protein, and, in cases where thepolynucleotide construct is DNA, the team “expresses Fab” meanstranscription of DNA into mRNA and translation of the mRNA into the Fabprotein. In cases where the polynucleotide construct is DNA, a promotersequence recognized by RNA polymerase for transcription into mRNA ispreferably additionally contained. The promoter may be appropriatelyselected depending on the expression system to be used. For example, incases where an E. coli cell or a cell-free translation system derivedfrom E. coli is employed, examples of the promoter include promotersthat function in E. coli, such as T7 promoter, T3 promoter, SP6promoter, and endogenous promoters in the E. coli genome.

The polynucleotide construct may be incorporated into a plasmid vector,phage vector, virus vector or the like. The type of the vector may beappropriately selected depending on the translation system and thescreening system used. The polynucleotide constructs described above andvectors containing them may be prepared by known genetic engineeringmethods described in Molecular Cloning (Cold spring Harbor LaboratoryPress, Cold spring Harbor (USA), 2001) and the like.

In the first embodiment and the second embodiment described above,either the Fab H chain-coding sequence or the Fab L chain-codingsequence may be placed upstream of the other. In cases where the Fab Hchain-coding sequence is placed upstream (in the 5′-side) and the Hchain is first translated and kept in the vicinity, followed by itspairing with the L chain immediately after completion of translation ofthe L chain, the risk of pairing between L chains, which is said tooccur more easily than pairing between H chains, can be reduced, whichis advantageous. On the other hand, in cases where the Fab Lchain-coding sequence is placed upstream (in the 5′-side) and the Lchain is first translated and kept in the vicinity, followed by itspairing with the H chain immediately after completion of translation ofthe H chain, the risk of occurrence of aggregation of H chains, whichare said to generally have higher risk of aggregation because of worsephysical properties than L chains, can be reduced.

<Method for Producing Fab>

The method of the present invention for producing Fab comprises the stepof introducing the polynucleotide construct described above into acell-free translation system containing ribosomes, to produce Fab.Examples of the cell-free translation system include cell-freetranslation systems obtained from cells of E. coli, yeasts, mammaliancells and the like, and the cell-free translation system is preferablyderived from E. coli.

The cell-free translation system may be either a cell extract obtainedby extracting a fraction containing ribosomes from cells, or areconstruction-type cell-free translation system constructed fromfactors purified individually.

A cell extract-type cell-free translation system is generally preparedby homogenizing cells and removing unwanted substances byultracentrifugation at about 30,000 g to obtain a cell extract calledS30, which is then subjected to an appropriate treatment. As a startingmaterial for S30, cells of various organisms have been tested so far,and 3 types of materials, E. coli (Zubay, Annual Review of Genetics(1973) vol. 7, p. 267-287), wheat germ (Roberts, et al., Proc Natl AcadSci USA (1973) vol. 70, p. 2330-2334) and rabbit reticulocytes (Pelham,et al., Eur. J. Biochem. (1976) vol. 67, p. 247-256) are commonly usedat present. In cases where E. coli is used as a starting material,various mutant strains may be used depending on the purpose, forpreparing S30. For example, when one wants to stabilize lineardouble-stranded DNA to be used as a template in the reaction liquid, theSL119 strain (Lesley, et al., J. Biol. Chem. (1991) vol. 266, p.2632-2638), which is a mutant strain deficient in RecD, a subunit of theRecBCD complex (Exonuclease V), may be used.

Examples of the reconstruction-type cell-free translation systemconstituted by factors purified individually include the PURE systemdescribed in JP 2003-102495 A and JP 2008-271903 A. Since thisreconstruction-type cell-free translation system can preventcontamination of nuclease and protease more easily than a cell-freetranslation system using a cell extract, the efficiency of translationof mRNA into the polypeptide can be increased.

In cases of the CIS display, double-stranded DNA, which is relativelystable in a cell extract, is used as a gene medium, so that a cellextract obtained by extracting a fraction containing ribosomes fromcells can also be used.

In cases of the ribosome display, there is the risk of degradation ofRNA as a gene medium by ribonuclease contained in a large amount in thecell extract, so that a reconstruction-type cell-free translation systemconstituted by factors purified individually is preferred. Such ribosomedisplay using the PURE system is called “PURE ribosome display (PRD)”.

The factors can be obtained by purification from extracts of variouscells. Examples of the cells from which the factors are purified includeprokaryotic cells and eukaryotic cells. Examples of the prokaryoticcells include E. coli cells, extreme thermophile cells and Bacillussubtilis cells. Examples of the eukaryotic cells include yeast cells,plant cells, insect cells and mammalian cells.

It is more preferred to individually purify ribosomes and other factorsto prepare the reconstruction-type cell-free translation system.

A ribosome is a huge complex constituted by ribosomal RNA and variousribosomal proteins, and composed of 2 subunits, that is, the largesubunit and the small subunit. The ribosome and the subunitsconstituting it can be separated from each other by sucrose densitygradient or the like, and their sizes are represented by thesedimentation coefficient. More specifically, in prokaryotes, theribosome and the subunits constituting it have the following sizes.Since prokaryotes such as E. coli can be easily prepared by large-scaleculture, a prokaryote such as E. coli is preferred as the organism fromwhich ribosomes are to be prepared in a large amount.

Ribosome (70S)=large subunit (50S)+small subunit (30S)

Molecular weight: about 2.5×10⁶ about 1.6×10⁶ about 0.9×10⁶

Still more specifically, it is known that each of the 50S subunit andthe 30S subunit is constituted by the following components.

50S subunit:

-   -   34 types of proteins L1 to L34 (ribosomal proteins)    -   23S RNA (about 3200 nucleotides)    -   5S RNA (about 120 nucleotides)

30S subunit:

-   -   21 types of proteins S1 to S21 (ribosomal proteins)    -   16S RNA (about 1540 nucleotides)        That is, each subunit can be isolated as a complex composed of        these components. Further, a ribosome can be isolated as a        complex of the subunits. A purified ribosome means, for example,        in cases of a ribosome derived from a prokaryote, the complex        purified as a 70S ribosome composed of the large and small        subunits, or a complex formed by mixing the 50S subunit and the        30S subunit together which were individually purified.

On the other hand, in eukaryotes, the ribosome and the subunitsconstituting it have the following sizes: ribosome (80S)=large subunit(60S)+small subunit (40S). Therefore, in cases where the cell-freetranslation system is to be constituted with ribosomes derived from aeukaryote, ribosomes purified as 80S ribosomes can be used.

Examples of the factors other than ribosomes to be added to thecell-free translation system include the following factors. Thesefactors are not limited to factors derived from prokaryotes such as E.coli, and factors derived from eukaryotes may also be used. Thesefactors and the methods for purifying these factors are known (JP2003-102495 A).

Initiation Factors (IFs)

Elongation factors (EFs)

Aminoacyl-tRNA synthetase

Methionyl-tRNA transformylase (MTF)

Although a releasing factor may also be contained, a releasing factor ispreferably not contained, for stable maintenance of the complex betweenthe protein and the polynucleotide on the ribosome.

The initiation factor is a factor that is indispensable for, or thatremarkably promotes, formation of an initiation complex. As initiationfactors derived from E. coli, IF1, IF2 and IF3 are known (Claudio O etal. (1990) Biochemistry, vol. 29, p. 5881-5889). The initiation factorIF3 promotes dissociation of the 70S ribosome into the 30S subunit andthe 50S subunit, which is a step necessary for initiation oftranslation, and inhibits insertion of tRNAs other than foimylmethionyltRNA into the P site upon formation of the initiation complex. Theinitiation factor IF2 is bound to formylmethionyl tRNA and carries thefoimylmethionyl tRNA to the P site of the 30S ribosome subunit, to forman initiation complex. The initiation factor IF1 promotes the functionsof the initiation factors IF2 and IF3. For example, in cases whereinitiation factors derived from E. coli are used, they may be used at0.01 μM to 300 μM, preferably 0.04 μM to 60 μM.

As elongation factors derived from E. coli, EF-Tu, EF-Ts and EF-G areknown. As the elongation factor EF-Tu, two types, the GTP type and theGDP type, are known, and the GTP type is bound to an aminoacyl-tRNA andcarries the aminoacyl-tRNA to the A site of the ribosome. When EF-Tu isreleased from the ribosome, GTP is hydrolyzed to cause its conversion tothe GDP type (Pape T et al, (1998) EMBO J, vol. 17, p. 7490-7497). Theelongation factor EF-Ts is bound to EF-Tu (GDP type), to promote itsconversion to the GTP type (Hwang Y W et al. (1997) Arch. Biochem.Biophys., vol. 348, p. 157-162). The elongation factor EF-G promotes thetranslocation reaction after the peptide bond formation reaction in theprocess of the peptide chain elongation (Agrawal R K et al, (1999) Nat.Struct. Biol., vol. 6, p. 643-647, Rodnina M W. et al, (1999) FEMSMicrobiology Reviews, vol. 23, p. 317-333). For example, in cases whereelongation factors derived from E. coli are used, they may be used at0.005 μM to 200 μM, preferably 0.02 μM to 50 μM.

The aminoacyl-tRNA synthetase (AARS) is an enzyme that covalently bindsan amino acid to a tRNA in the presence of ATP to synthesize anaminoacyl-tRNA, and there are specific relationships between amino acidsand aminoacyl-tRNA synthetases (Francklyn C et al, (1997) RNA, vol. 3,p. 954-960; Proteins, Nucleic Acids and Enzymes, vol. 39, p. 1215-1225(1994)). For example, in cases where aminoacyl-tRNA synthetases derivedfrom E. coli are used, they may be used at 0.01 μg/ml to 10,000 μg/ml,preferably 0.05 μg/ml to 5,000 μg/ml.

Methionyl-tRNA transformylase (MTF) is an enzyme that synthesizesN-formylmethionyl (fMet) initiation tRNA, wherein a formyl group isattached to the amino group of methionyl initiation tRNA, in proteinsynthesis in prokaryotes. That is, methionyl-tRNA transformylasetransfers the formyl group of FD to the amino group of the methionylinitiation tRNA corresponding to the initiation codon, to form thefMet-initiation tRNA (Ramesh V et al, (1999) Proc. Natl. Acad. Sci. USA,vol. 96, p. 875-880). The attached formyl group is recognized by theinitiation factor IF2 and acts as the initiation signal for proteinsynthesis. The protein synthesis system in the cytoplasm of eukaryotesdoes not have MTF, but the protein synthesis systems in mitochondria andchloroplasts in eukaryotes have MTF. Preferred examples of the MTFinclude those derived from E. coli, such as the one obtained from the E.coli K12 strain. In cases where MTF derived from E. coli is used, it maybe used at, for example, 100 U/ml to 1,000,000 U/ml, preferably 500 U/mlto 400,000 U/ml. The activity herein is defined as 1 U when 1 pmol offMet-initiation tRNA is formed in 1 minute. The formyl donor (FD) as thesubstrate of MTF may be used at, for example, 0.1 μg/ml to 1000 μg/ml,preferably 1 μg/ml to 100 μg/ml.

In cases where the polynucleotide to be added to the reaction liquid isDNA, RNA polymerase for transcription into mRNA may be contained. Morespecifically, the following RNA polymerases may be used. These RNApolymerases are commercially available.

T7 RNA polymerase

T3 RNA polymerase

SP6 RNA polymerase

In cases where T7 RNA polymerase is used, it may be used at 0.01 μg/mlto 5000 μg/ml, preferably 0.1 μg/ml to 1000 μg/ml. Further, in caseswhere a reconstruction-type cell-free translation system is used in CISdisplay for increasing the efficiency of formation of the complexbetween Fab and DNA, endogenous RNA polymerase purified from E. coli maybe added as described in Nucleic Acid Research 2010, vol. 38, No. 13,e141. Further, a transcription termination factor rho protein purifiedfrom E. coli may also be added as described in Nucleic Acid Research1988, vol. 16, No. 14, 6493.

The cell-free translation system may also contain, in addition to thefactors for transcription and translation, auxiliary components.Examples of the auxiliary components include the following components.

Enzymes for regeneration of energy in the reaction system:

-   -   creatine kinase;    -   myokinase;    -   nucleoside diphosphate kinase; and the like.

Enzymes for degradation of inorganic pyrophosphate produced intranscription/translation:

Inorganic pyrophosphatase; and the like.

The above enzymes may be used at, for example, 0.01 μg/ml to 2000 μg/ml,preferably 0.05 μg/ml to 500 μg/ml.

The cell-free translation system preferably contains amino acids,nucleoside triphosphates, tRNAs and salts. Further, in cases where thereaction system is derived from a prokaryotic cell such as E. coli, thecell-free translation system preferably contains the methionyl-tRNAtransformylase and 10-formyl 5,6,7,8-tetrahydrofolate (FD).

As the amino acids, naturally-occurring amino acids as well asnon-naturally occurring amino acids may be used. These amino acids arecarried by tRNAs by the action of aminoacyl-tRNA synthetasesconstituting the cell-free translation system. Alternatively, the aminoacids may be preliminarily charged to tRNAs before addition into thecell-free translation system. The charging of an amino acid to tRNAherein means that tRNA is made to carry an amino acid such that theamino acid can be used in the translation reaction on a ribosome. Byadding non-naturally occurring amino acids in the presence of artificialaminoacyl synthetases that recognize the non-naturally occurring aminoacids, or by using tRNAs charged with non-naturally occurring aminoacids, the non-naturally occurring amino acids can be introduced intospecific codon sites of the protein. In cases where naturally occurringamino acids are used, they may be used at 0.001 mM to 10 mM, preferably0.01 mM to 2 mM.

As the tRNAs, tRNAs purified from cells of E. coli, yeast or the likemay be used. Artificial tRNAs wherein anticodons and/or other bases areartificially modified may also be used (Hohsaka, et al. (1996) J. Am.Chem. Soc., vol. 121, p. 34-40, Hirao I et al (2002) Nat. Biotechnol.,vol. 20, p. 177-182). For example, by charging a non-naturally occurringamino acid to a tRNA having CUA as the anticodon, the UAG codon, whichis originally a stop codon, can be translated to the non-naturallyoccurring amino acid. Further, by using an artificial aminoacyl-tRNAwherein a non-naturally occurring amino acid is charged to a tRNA havinga 4-base codon as the anticodon, the non-naturally occurring 4-basecodon can be translated to the non-naturally occurring amino acid(Hohsaka et al. (1999) J. Am. Chem. Soc., vol. 121, p. 12194-12195). Asthe method for preparing such an artificial tRNA, a method using RNA mayalso be used (Japanese Translated PCT Patent Application Laid-open No.2003-514572). By such methods, a protein having site-specificallyintroduced non-naturally occurring amino acids can be synthesized. Incases where an E. coli tRNA mixture is used, the tRNAs may be used at,for example, 0.1 A₂₆₀/ml to 1000 A₂₆₀/ml, preferably 1 A₂₆₀/ml to 500A₂₆₀/ml.

The reconstruction-type cell-free translation system can be prepared byadding the factors to a buffer with a constant pH of 7 to 8, which issuitable for transcription and translation. Examples of the bufferinclude potassium phosphate buffer (pH 7.3) and Hepes-KOH (pH 7.6). Incases where Hepes-KOH (pH 7.6) is used, it may be used, for example, at0.1 mM to 200 mM, preferably 1 mM to 100 mM.

The cell-free translation system may also contain salts for the purposesof protecting the factors and maintaining the activities. Specificexamples of the salts include potassium glutamate, potassium acetate,ammonium chloride, magnesium acetate, magnesium chloride and calciumchloride. These salts are used usually at 0.01 mM to 1000 mM, preferablyat 0.1 mM to 200 mM.

The cell-free translation system may also contain otherlow-molecular-weight compounds as substrates for enzymes, and/or for thepurpose of increasing or maintaining the activities. Specific examplesof the compounds which may be added to the cell-free translation systeminclude substrates such as nucleoside triphosphates (e.g., ATP, GTP, CTPand UTP); polyamines such as putrescine and spermidine; reducing agentssuch as dithiothreitol (DTT); and substrates for regeneration of energy,such as creatine phosphate. These low molecular weight compounds may beused usually at 0.01 mM to 1000 mM, preferably at 0.1 mM to 200 mM.

The cell-free translation system may be prepared according to thespecific compositions described in Shimizu et al. (Shimizu et al., Nat.Biotechnol. (2001) vol. 19, p. 751-755; Shimizu et al., Methods (2005)vol. 36, p. 299-304) or Ying et al. (Ying et al., Biochem. Biophys. Res.Commun. (2004) vol. 320, p. 1359-1364). However, the concentration ofeach factor may be increased or decreased as appropriate depending onthe specific activity and/or the purpose of the purified factor. Forexample, in cases where the energy consumption is high, ATP may beincreased. Further, depending on the codon usage in the mRNA to betranslated, specific tRNAs may be added.

In cases where the protein is one which hardly forms a higher orderstructure, a group of proteins called molecular chaperones may becontained. Specific examples of the molecular chaperones which may beadded to the cell-free translation system include Hsp100, Hsp90, Hsp70,Hsp60, Hsp40, Hsp10 and low molecular weight Hsp, and their homologues;and trigger factors in E. coli. Molecular chaperones are proteins knownto assist formation of higher order structures of proteins and preventaggregation of proteins in the cell (Bukau and Horwich, Cell (1998) vol.92, p. 351-366; Young et al., Nat. Rev. Mol. Cell Biol (2004) vol. 5, p.781).

When Fab is expressed, disulfide bonds are formed in the molecule, sothat the oxidation-reduction potential of the reaction liquid isimportant. Therefore, DTT as a reducing agent may be removed from thereaction liquid, and/or a cell-free translation system supplemented withglutathione may be used. Further, a cell-free translation systemsupplemented with an enzyme(s) that promote(s) disulfide bonding and/orcorrect the bonds may be used. Specific examples of such enzymes includeprotein disulfide isomerase (PDI) present in ER of eukaryotic cells, andDsbA and DsbC in E. coli.

By introducing the polynucleotide construct into the cell-freetranslation system as described above to perform translation reaction,Fab can be obtained. The obtained Fab may be purified using an affinitycolumn and/or the like.

<Screening Method>

The screening method of the present invention comprises the steps of:(i) introducing a polynucleotide construct encoding a Fab library into acell-free translation system to synthesize Fabs, and displaying thesynthesized Fabs on the polynucleotides encoding the Fabs; (ii) bringingthe Fabs displayed on the polynucleotides into contact with an antigen;(iii) selecting a Fab of interest that reacts with the antigen; and (iv)amplifying the polynucleotide encoding the Fab of interest.

In the cell-free translation system as described above, Fab is displayedon a ribosome and/or mRNA based on the given genetic information. Inribosome display, the polypeptide containing Fab is kept accompanied bythe genetic information (mRNA) encoding it by stalling of the ribosomeon mRNA due to the presence of a ribosome stall sequence at the 3′-end.That is, a complex of the 3 factors, mRNA-ribosome-polypeptide is famed.On the other hand, in mRNA display utilizing puromycin or a derivativethereof, the C-terminus of the polypeptide containing Fab is covalentlybound to the puromycin or a derivative thereof due to the presence ofthe puromycin or a derivative thereof at the 3′-end of thepolynucleotide construct (mRNA), and the link between the polypeptidecontaining Fab and the mRNA encoding it is maintained. Further, in CISdisplay, the polypeptide containing Fab is expressed as a fusion proteinwith a DNA-binding protein, and the link between the polypeptidecontaining Fab and the DNA encoding it is maintained by binding of theDNA-binding protein to its target sequence on the DNA. In either method,by selecting Fabs bound to the antigen and recovering mRNAs boundthereto, the genetic information of the Fabs is also recovered. From themRNAs in the complexes each containing a Fab bound to the target antigenand mRNA, cDNAs are synthesized, and the cDNAs are then amplified byPCR. Transcription/translation reaction is then performed again. Byrepeating these steps, antibodies against the antigen of interest can beobtained.

The above process is described below more concretely.

The size of the Fab gene library is usually not less than 1×10⁸,preferably not less than 1×10⁹, more preferably not less than 1×10¹⁰,still more preferably not less than 1×10¹².

The Fab library expressed from the Fab gene library is brought intocontact with the target substance (antigen), and Fabs that bind to thetarget substance are selected from the Fab library, followed byamplification of the polynucleotides encoding the Fabs.

For selection of the Fabs bound to the target substance, the Fabs boundto the target substance need to be screened from numerous Fabs that arenot bound to the target substance. This is carried out by the knownmethod called panning (Coomber (2002) Method Mol. Biol., vol. 178, p.133-145). A basic protocol for panning is as follows.

(1) The Fab library is brought into contact with the target substance.The target substance may be bound to a carrier such as a bead, plate orcolumn, and a sample containing the complex between the Fab and thepolynucleotide may be brought into contact therewith (solid-phaseselection). Alternatively, selection in the liquid phase may be carriedout wherein, for example, the target substance is biotinylated and boundto the Fab, and the complex between the target substance and the Fab isrecovered with streptavidin magnetic beads. Further, the solid-phaseselection and the liquid-phase selection may be used in combination. Bycarrying out a plurality of rounds of screening such that thesolid-phase selection is first performed and the liquid-phase selectionis performed later, high-affinity antibodies can be efficientlyselected.

(2) Other Fabs which are unbound to the target substance and containedin the library are removed. For example, such Fabs can be removed bywashing. The washing can be carried out with a washing liquid which isused for washing after usual antigen-antibody reaction, and, in caseswhere screening is carried out for obtaining a Fab having higheraffinity than the parent Fab, the washing is preferably carried outunder conditions where the bond between the parent Fab and the antigenis maintained while weaker bonds are removed.

(3) The Fabs that were not removed, that is, the Fabs that werespecifically bound to the target substance, are recovered.

(4) The operations of (1) to (3) are repeated a plurality of times asrequired.

In cases of ribosome display, mRNA display or CIS display, when theseries of steps are repeated, the polynucleotide in the recoveredcomplex containing the polypeptide-polynucleotide is amplified beforeStep (1). For example, mRNA can be amplified by RT-PCR. By the RT-PCR,DNA is synthesized using the mRNA as a template. The DNA may betranscribed into mRNA again to use it for formation of the complex.FIGS. 7 and 8 are schematic diagrams showing examples of the screeningmethod for Fab, wherein the series of operations, that is, transcriptionof DNAs into mRNAs; translation of the mRNAs to produce Fabs; selectionof Fabs bound to an antigen; and recovery and amplification of the mRNAstherefrom; are shown.

By the above operations, polynucleotides encoding Fabs that specificallybind to the antigen of interest are enriched. The amino acid sequenceinformation of the Fab of interest can be identified by analyzing thesequence of the obtained polynucleotide.

In cases where an antibody against the antigen of interest has alreadybeen obtained and the sequences of the CDRs of the antibody are known,an antibody having even higher affinity can be obtained by using thescreening method of the present invention.

That is, the present invention provides a method for screening Fab,which method comprises the steps of:

(I) providing a plurality of types of the polynucleotide construct ofthe present invention, in each of which the Fab first chain-codingsequence or the Fab second chain-coding sequence encodes an amino acidsequence comprising a single amino acid substitution at a singleposition in a CDR in the amino acid sequence of the Fab first chain orthe Fab second chain of the parent antibody, such that single amino acidsubstitutions are contained for a plurality of positions in the CDRs ofthe Fab first chain and the Fab second chain;

(II) carrying out first screening wherein the Steps (i) to (iv) arerepeated using the plurality of types of the polynucleotide construct,to screen a plurality of high-affinity Fabs;

(III) analyzing single amino acid substitutions at respective positionsin the CDRs of the Fab first chain and the Fab second chain in theplurality of Fabs selected in the first screening step;

(IV) providing the polynucleotide construct of the present inventionwherein the Fab first chain-coding sequence and the Fab secondchain-coding sequence encode amino acid sequences comprisingcombinations of the single amino acid substitutions identified in thefirst screening at the respective positions in the CDRs of the Fab firstchain and Fab second chain sequences of the parent antibody; and

(V) carrying out second screening wherein the Steps (i) to (iv) arerepeated using the polynucleotide construct, to screen a high-affinityFab.

The “plurality” in Step (I) is not restricted as long as it is 2 ormore, and the “plurality” preferably means the number of all amino acidsin the CDRs.

The screening method for increasing the affinity of Fab is concretelydescribed below.

However, the screening method of the present invention is not limited tothis embodiment.

First, the amino acid sequence (parent sequence) of an antibody (parentantibody) against the antigen of interest is provided. Subsequently, asshown in FIG. 24, polynucleotide constructs of the present inventioneach containing: an H chain-coding sequence having the same amino acidsequence as in the parent antibody except that one of the total aminoacids in the CDRs of the H chain (8 amino acids in CDR-H1, 11 aminoacids in CDR-H2 and 12 amino acids in CDR-H3—a total of 31 amino acids)has a single amino acid substitution (which preferably allows appearanceof all 20 naturally-occurring amino acids including the parent aminoacid); and the L chain-coding sequence of the parent antibody; areprovided in a number equal to the number of all amino acids (31 aminoacids in total) in the CDRs of the H chain (H-chain library).

Further, polynucleotide constructs each containing: an L chain-codingsequence having the same amino acid sequence as in the parent antibodyexcept that one of the total amino acids in the CDRs of the L chain (7amino acids in CDR-L1, 6 amino acids in CDR-L2 and 6 amino acids inCDR-L3—a total of 19 amino acids) has a single amino acid substitution(which preferably allows appearance of all 20 naturally-occurring aminoacids including the parent amino acid); and the H chain-coding sequenceof the parent antibody; are provided in a number equal to the number ofall amino acids (19 amino acids in total) in the CDRs of the L chain(L-chain library).

Using the above H-chain library and the L-chain library as a primarylibrary, Steps (i) to (iv) are repeated to carry out screening ofhigh-affinity antibodies (first screening).

Since the polynucleotide sequences encoding the high-affinity antibodiesare enriched by the first screening, the plurality of enriched sequencesare analyzed to select amino acid substitutions frequently observed atrespective positions in the CDRs of the H chain and the L chain as aminoacid substitutions preferred for increasing the affinity.

Although sequence analysis of the polynucleotides can be carried out byconventional sequencing, a next-generation sequencer is preferably usedsince cloning of the Fab-coding sequences amplified by the screening isnot necessary; large-scale comprehensive sequence analysis is possible;and the time required for screening can be largely reduced.

There team “next-generation sequencer” herein is used in contrast to thefirst-generation sequencer represented by the fluorescent capillarysequencer utilizing the Sanger sequencing method. This term actuallyincludes various apparatuses and techniques, and invention of variousmodes of such a sequencer can be expected also in the future (Mardis,Annu. Rev. Genom. Human Genet. (2008) vol. 9, p. 387-402; Persson etal., Chem. Soc. Rev. (2010) vol. 39, p. 985-999).

In contrast to the first-generation sequencer which is generally usedfor sequencing of DNA cloned using a vector-host system, thenext-generation sequencer enables rapid determination of DNA sequencesin samples without cloning of DNA using a vector-host system, while DNAsamples having various sequences can be analyzed therewith.

In determination of DNA sequences using a first-generation sequencer,the DNA sequences of individual clones are determined by the followingprocess:

(i) incorporation of each DNA fragment into a vector such as a plasmid,and transformation of a host such as E. coli with the resultant;

(ii) cloning by isolation of host colonies;

(iii) culture of clones in a number dependent on the scale of theanalysis, and extraction of their plasmids;

(iv) sequencing reaction by PCR or the like using each plasmid or thelike as a template; and

(v) separation, detection and analysis of the sequencing reactionproduct by capillary electrophoresis and/or the like. In cases where theDNA sequences of a large number of clones are to be determined at once,the reactions/treatments in the Steps (ii) to (v) after cloning need tobe carried out separately for each clone, so that the amount of laborrequired is proportional to the number of clones to be analyzed.

On the other hand, in DNA sequencing using a next-generation sequencer,DNA fragments having various sequences are individually cloned on a vastnumber of analytical spots on a substrate for analysis and subjected tomassively parallel sequence analysis, by application of an amplificationtechnique such as single-molecule PCR including Emulsion PCR and BridgePCR, or a highly sensitive detection technique such as single moleculeobservation. Therefore, the sequences of, for example, 10⁶ to 10⁹ clonesfamed on a single substrate for analysis can be determined by a singlereaction/treatment irrespective of the number of clones to be analyzed.

The next-generation sequencer which is most common at present is basedon a principle in which sequential DNA synthesis with DNA polymerase orDNA ligase is used for massively parallel determination of nucleotidesequences by optical detection of fluorescence, luminescence or thelike. Examples of known methods using DNA polymerase include GS FLX(Roche Diagnostics) and Solexa (Illumina), and examples of known methodsusing DNA ligase include SOLiD (Applied Biosystems) and Polonator (DoverSystems). Further, HeliScope (Helicos Biosciences), SMRT (PacificBiosciences) and the like are known, which determine nucleotidesequences by single-molecule real-time observation wherein a singlemolecule of DNA is used as a template for synthesis of DNA by DNApolymerase and the reaction for each single base is optically detectedwith fluorescence, luminescence or the like. Other reported examplesinclude a principle called Post-light sequencing, wherein massivelyparallel determination of nucleotide sequences is carried out by adetection method other than optical detection of fluorescence,luminescence or the like. Examples of methods that correspond to thisprinciple include Ion Torrent Systems, wherein a semiconductor (CMOS)chip is used to detect hydrogen ions (pH change) released uponincorporation of each base by DNA polymerase, in order to determinenucleotide sequences. Other known examples include Nanopore (OxfordNanopore Technologies).

The amino acid sequences obtained by the first screening, containing thecombinations of single amino acid substitutions in the amino acids ofCDRs in the H chain and the L chain, are used as the secondary library.For example, in cases where the first screening suggested that the firstamino acid is preferably substituted to Ala or Thr and the second aminoacid is preferably substituted to Ile or Leu in the first CDR of the Hchain (H1), the library to be used in the second screening, that is, thesecondary library, is prepared by designing nucleotide sequences suchthat the first amino acid is Ala or Thr and the second amino acid is Ileor Leu, and other amino acids also show preferred combinations of aminoacids obtained in the first screening. It should be noted that thepreferred mutant amino acids obtained in the first screening and theamino acids in the parent sequence may be combined to provide thesecondary library.

Using the thus obtained secondary library, Steps (i) to (iv) arerepeated to carry out the second screening. By this, many types ofantibodies having remarkably improved affinity to the antigen relativeto the parent antibody can be obtained. The remarkable improvement ofaffinity to the antigen relative to the parent antibody can be analyzedby SPR, ELISA or the like.

The polynucleotide construct of the present invention may be used as acomponent of a kit for production and/or screening of Fabs.

The principle of two-step affinity maturation in the Ymacs method of thepresent invention, which comprises the steps of searching of potentiallybeneficial single amino acid substitutions from the primary library andsearching of the optimal combination of these beneficial single aminoacid substitutions from the secondary library, can be applied toimprovement of the affinity of not only Fab fragments of antibodies butalso scFv, full-length antibodies and other target-binding proteins tothe target, in combination with a wide range of protein display systems.

That is, the present invention provides a method for maximizing theaffinity of a target-substance-binding protein to a target substance(method for obtaining a mutant-type target-substance-binding proteinhaving improved affinity to a target substance), which method comprisesthe steps of:

(I) constructing single-position libraries wherein one amino acid amongall amino acid positions constituting a target-substance-binding site ina target-substance-binding protein is randomized to all the 20 types ofnaturally-occurring amino acids, to provide as many single-positionlibraries as the number of the all amino acid positions;

(II) constructing a primary library by integrating all of, or anappropriate unit of, these single-position libraries (preparation ofprimary-library polynucleotide constructs);

(III) selecting the primary library using a protein display system basedon the affinity to a target;

(IV) determining the polynucleotide sequence information of the selectedsample of the primary library obtained in (III);

(V) extracting single amino acid substitutions frequently observed(preferably 2 or more times) in the nucleotide sequence information;

(VI) constructing a secondary library comprising combinations of thefrequently observed single amino acid substitutions (preparation ofsecondary-library polynucleotide constructs); and

(VII) selecting the secondary library using a protein display systembased on the affinity to a target.

In cases where the primary library is constructed by integration of anappropriate unit in Step (II), the single-position libraries constructedin Step (I) may be divided into 2 or more groups, to provide 2 or moretypes of primary libraries. Further, the secondary library constructedin (VI) may contain, in addition to the frequently observed single aminoacid substitutions, the parent amino acids encoded by the parentantibody.

Examples of the target-substance-binding protein herein includeantigen-binding proteins and cytokines.

Examples of the antigen-binding protein include Fab, scFv andfull-length antibodies, and the antigen-binding protein may also be aprotein that is not classified as an antibody, as long as itspecifically binds to an antigen.

Further, a mutation(s) may also be introduced into a cytokine forincreasing its binding capacity to a receptor.

The protein display system is not limited as long as the system allowsassociation of proteins with the polynucleotides encoding them, andexamples of the protein display system include not only theabove-described cell-free systems such as ribosome display, mRNA displayand CIS display, but also other protein display systems such as phagedisplay (Non-patent Document 1), bacterial surface display (Jose, Appl.Microbiol. Biotechnol. (2006) vol. 69, p. 607-614), yeast surfacedisplay (Feldhaus et al., Nat. Biotechnol. (2003) vol. 21, p. 163-70),and cell surface display with a higher eukaryote (Horlick,WO2008/103475). The part other than the coding region for the targetsubstance-binding protein in the single-position library polynucleotidemay have the same constitution as a known vector for each proteindisplay. The coding region for the target substance-binding proteincontained in the library polynucleotide may encode either thefull-length of the target substance-binding protein or a part of thetarget substance-binding protein wherein the target substance-bindingsite is contained.

A more concrete description is given below by way of an example.

For example, in cases where the target substance-binding site in thetarget substance-binding protein (wild type) is a region composed of 10amino acids, a total of 10 types of single-position libraries in each ofwhich 20 amino acids appear at a single position among the 1st to 10thpositions are provided.

These 10 types of single-position libraries are combined to prepare aprimary library, and this library is subjected to the selection based onthe affinity to the target substance. More specifically, the Steps (I′)to (iv′) described below are repeated to concentrate sequences havinghigh affinity to the target substance (first screening):

(i′) the step of introducing the library into a protein display systemto allow expression of the target substance-binding proteins, anddisplaying the target substance-binding proteins on the polynucleotidesencoding the proteins;

(ii′) the step of bringing the target substance-binding proteins intocontact with a target substance;

(iii′) the step of selecting target substance-binding proteins ofinterest that react with the target substance; and

(iv′) the step of amplifying the polynucleotides encoding the targetsubstance-binding proteins of interest.

These steps may be carried out in the same manner as the operations innormal protein display.

In Step (ii′), the target substance may be bound to a carrier such as abead, plate or column, and a sample containing complexes between thetarget substance-binding proteins and the polynucleotides may be broughtinto contact therewith (solid-phase selection). Alternatively, selectionin the liquid phase may be carried out wherein, for example, the targetsubstance is biotinylated and bound to the target substance-bindingproteins, and the complexes between the target substance and the targetsubstance-binding proteins are recovered with streptavidin magneticbeads. Further, the solid-phase selection and the liquid-phase selectionmay be used in combination. By carrying out a plurality of rounds ofscreening such that the solid-phase selection is first performed and theliquid-phase selection is performed later, high-affinity proteins can beefficiently selected.

For removing non-specific binding, a washing operation is preferablycarried out between (ii′) and (iii′). The washing is preferably carriedout under conditions where the bond between the wild-type targetsubstance-binding protein and the target substance is maintained whileweaker bonds are removed.

Since the polynucleotide sequences encoding proteins having highaffinity to the target substance are enriched by the first screening,the sequences of the plurality of enriched target substance-bindingsites are analyzed to select amino acid substitutions frequentlyobserved at respective positions in the CDRs of the H chain and the Lchain as amino acid substitutions preferred for increasing the affinity.

Although sequence analysis of the polynucleotides may be carried out byconventional sequencing, a next-generation sequencer is preferably usedsince cloning of the coding sequences for the target substance-bindingproteins amplified by the screening is not necessary; large-scalecomprehensive sequence analysis is possible; and the time required forscreening can be largely reduced.

There team “next-generation sequencer” herein is used in contrast to thefirst-generation sequencer represented by the fluorescent capillarysequencer utilizing the Sanger sequencing method. This term actuallyincludes various apparatuses and techniques, and invention of variousmodes of such a sequencer can be expected also in the future (Mardis,Annu. Rev. Genom. Human Genet. (2008) vol. 9, p. 387-402; Persson etal., Chem. Soc. Rev. (2010) vol. 39, p. 985-999).

In contrast to the first-generation sequencer which is generally usedfor sequencing of DNA cloned using a vector-host system, thenext-generation sequencer enables rapid determination of DNA sequencesin samples without cloning of DNA using a vector-host system, while DNAsamples having various sequences can be analyzed therewith.

In determination of DNA sequences using a first-generation sequencer,the DNA sequences of individual clones are determined by the followingprocess:

(i) incorporation of each DNA fragment into a vector such as a plasmid,and transformation of a host such as E. coli with the resultant;

(ii) cloning by isolation of host colonies;

(iii) culture of clones in a number dependent on the scale of theanalysis, and extraction of their plasmids;

(iv) sequencing reaction by PCR or the like using each plasmid or thelike as a template; and

(v) separation, detection and analysis of the sequencing reactionproduct by capillary electrophoresis and/or the like. In cases where theDNA sequences of a large number of clones are to be determined at once,the reactions/treatments in the Steps (ii) to (v) after cloning need tobe carried out separately for each clone, so that the amount of laborrequired is proportional to the number of clones to be analyzed.

On the other hand, in DNA sequencing using a next-generation sequencer,DNA fragments having various sequences are individually cloned on a vastnumber of analytical spots on a substrate for analysis and subjected tomassively parallel sequence analysis, by application of an amplificationtechnique such as single-molecule PCR including Emulsion PCR and BridgePCR, or a highly sensitive detection technique such as single moleculeobservation. Therefore, the sequences of, for example, 10⁶ to 10⁹ clonesfamed on a single substrate for analysis can be determined by a singlereaction/treatment irrespective of the number of clones to be analyzed.

The next-generation sequencer which is most common at present is basedon a principle in which sequential DNA synthesis with DNA polymerase orDNA ligase is used for massively parallel determination of nucleotidesequences by optical detection of fluorescence, luminescence or thelike. Examples of known methods using DNA polymerase include GS FLX(Roche Diagnostics) and Solexa (Illumina), and examples of known methodsusing DNA ligase include SOLiD (Applied Biosystems) and Polonator (DoverSystems). Further, HeliScope (Helicos Biosciences), SMRT (PacificBiosciences) and the like are known, which determine a nucleotidesequence by single-molecule real-time observation wherein a singlemolecule of DNA is used as a template for synthesis of DNA by DNApolymerase and the reaction for each single base is optically detectedwith fluorescence, luminescence or the like. Other reported examplesinclude a principle called Post-light sequencing, wherein massivelyparallel determination of nucleotide sequences is carried out by adetection method other than optical detection of fluorescence,luminescence or the like. Examples of methods that correspond to thisprinciple include Ion Torrent Systems, wherein a semiconductor (CMOS)chip is used to detect hydrogen ions (pH change) released uponincorporation of each base by DNA polymerase, in order to determinenucleotide sequences. Other known examples include Nanopore (OxfordNanopore Technologies).

By comprehensively analyzing the sequences obtained by the firstscreening using a next-generation sequencer, the sequences can be usedin the second screening without cloning (cloning into a vector followedby expression of the individual sequences), so that the time requiredfor screening can be largely reduced, which is advantageous. However,the sequences may be partially cloned and subjected to investigation ofthe binding capacity for the purpose of, for example, confirmation ofthe efficiency of the first screening.

Subsequently, the single amino acid substitutions of the respectiveamino acids in the target substance-binding site, obtained by thesequence analysis in the first screening, are combined to prepare thesecondary library. For example, in cases where the first screeningsuggested that the first amino acid is preferably substituted to Ala orThr and the second amino acid is preferably substituted to Ile or Leu inthe target substance-binding site, the library to be used in the secondscreening, that is, the secondary library, is prepared by designingnucleotide sequences such that the first amino acid is Ala or Thr andthe second amino acid is Ile or Leu, and other amino acids also showpreferred combinations of amino acids obtained in the first screening.It should be noted that the preferred mutant amino acids obtained in thefirst screening and the amino acids in the parent sequence may becombined to provide the secondary library.

Using this secondary library, Steps (i′) to (iv′) are repeated to carryout the second screening. By this, many types of proteins havingremarkably improved affinity to the target substance relative to theparent protein can be obtained. The remarkable improvement of affinityto the target substance relative to the parent protein can be analyzedby SPR, ELISA or the like.

EXAMPLES

The present invention is described below more concretely by reference toExamples. However, the present invention is not limited to theembodiments below.

[Example 1] Preparation of Model Fab

As the framework of the Fab to be used in the cell-free Fab displaysystem, the combination of VL k subgroup I and VH subgroup III, which isone of the major antibody subclasses in human, excellent in expressionefficiency in E. coli (Kanappik et al., J Mol Biol. (2000) vol. 296, p.57-86) and reported to be effective in a cell-free scFv display system(Shibui et al., Appl Microbiol Biotechnol. (2009) vol. 84, p. 725-732),was selected. As a model Fab having the above-described framework to beused for confirmation of the performance of the cell-free Fab displaysystem, the human EGFR-2 (Her-2)-reactive Fab reported by Carter et al.(Carter et al., Proc Natl Acad Sci USA. (1992) vol. 89, p. 4285-4289)was selected to provide Fab-HH.

As another model Fab, Fab-TT was provided by substituting the CDR 1-3regions in the L chain and the H chain of Fab-HH by the sequence of theINFαR-reactive scFv discovered by the cell-free scFv display system byShibui et al. (Shibui et al., Biotechnol. Lett. (2009) vol. 31, p.1103-1110).

cDNAs encoding the VL domain and the VH domain of these 2 types of modelFabs were artificially synthesized. Further, cDNAs encoding the CLdomain and the CH1 domain were obtained by PCR using the genomic DNA ofa human cancer cell line as a template. The obtained cDNA fragments wereincorporated into an E. coli expression vector pTrc99A to construct pTrcFab-HH and pTrc Fab-TT as vectors to be used for bicistronic secretoryexpression of the model Fabs. Further, the original combinations of theL chain and the H chain were intentionally changed to provide Fab-HT(the combination of the Her-2-reactive L chain and the INFαR-reactive Hchain) and Fab-TH (the combination of the INFαR-reactive L chain and theHer-2-reactive H chain), and pTrc Fab-HT and pTrc Fab-TH which expressthese were constructed. In this process, a myc tag was attached to theC-terminus of the L chain, and a dual FLAG tag and a His tag wereconsecutively attached to the C-terminus of the H chain. A schematicview of the structure of the unit that bicistronically expresses Fab inthe pTrc Fab vector is shown in FIG. 9.

The nucleotide sequence of pTrc Fab-HH is shown in SEQ ID NO:1, and theamino acid sequence encoded thereby is shown in SEQ ID NOs:2 and 3. Thenucleotide sequence of pTrc Fab-TT is shown in SEQ ID NO:4, and theamino acid sequence encoded thereby is shown in SEQ ID NOs:5 and 6. Thenucleotide sequence of pTrc Fab-HT is shown in SEQ ID NO:7, and theamino acid sequence encoded thereby is shown in SEQ ID NOs:8 and 9. Thenucleotide sequence of pTrc Fab-TH is shown in SEQ ID NO:10, and theamino acid sequence encoded thereby is shown in SEQ ID NOs:11 and 12.

The E. coli DH5α strain having the pTrc Fab vector was cultured in LBmedium supplemented with 100 μg/ml ampicillin, and IPTG was addedthereto during the logarithmic phase at a final concentration of 0.1 mMto induce expression of the model Fab into the culture supernatant. Theculture supernatant was recovered 18 hours after the addition of IPTG,and the model Fab contained therein was bound to Ni-NTA agarose beads(QIAGEN) using the His tag attached at the C-terminus of the H chain.After washing the beads, elution was carried out with PBS supplementedwith imidazole at a final concentration of 250 mM. The eluted fractionwas dialyzed against PBS, to prepare a high-purity model Fab. Theconcentration of the model Fab was calculated based on the absorbance at280 nm under the assumption of 1 OD=1 mg/ml. The model Fab samples wereseparated by 5-20% gradient gel SDS-PAGE, followed by staining withCoomassie Blue. The result is shown in FIG. 10. It can be seen that thepurity of each prepared model Fab is sufficient.

[Example 2] Evaluation of Activities, Specificities and L Chain-H ChainInterdependencies of Model Fabs

Each of Her-2 and INFαR (R&D systems) as an antigen protein for themodel Fab was diluted with PBS to 1 μg/ml. The diluted antigen wasplaced on an ELISA plate, and immobilized on the plate by incubation at4° C. overnight. After washing the well, the model Fab was added theretoto bind the model Fab to the antigen. After washing the well, a1000-fold diluted HRP-labeled anti-FLAG antibody (Sigma) as a detectionantibody was added to the well, to bind the detection antibody to theFLAG tag attached to the model Fab. After washing the well, acolorimetric substrate for HRP was added thereto. When an appropriatedegree of coloring was achieved, a stop solution was added to the well,and the absorbance was measured at OD 405 nm. FIG. 11 shows the resultof titration of the 4 types of model Fabs over a wide range ofconcentration using Her-2 as the antigen protein. It can be seen thatFab-HH binds to Her-2 strongly (ED50=2 nM). FIG. 12 shows the resultobtained by bringing each of the 4 types of model Fabs at highconcentrations (7.1, 1.8, 2.9 and 5.8 μM, respectively, for HH, TT, HTand TH) into contact with each of the two types of antigen proteins. Itcan be seen that Fab-HH binds to Her-2 but does not bind to INFαR, andthat Fab-TT binds to INFαR but does not bind to Her-2. Further, sinceneither of the hybrid Fabs, Fab-HT and Fab-TH, binds to any of theantigens, it can be seen that the binding of Fab-HH to Her-2 and thebinding of Fab-TT to INFαR are L chain-H chain interdependent, and thata correct combination of the L chain and the H chain is necessary forbinding to an antigen protein.

[Example 3] Construction of Vector for Bicistronic Fab-PRD

A sequence having a very similar amino acid sequence to that of theabove-described Fab-HH and reported by Fellouse et al. (Fellouse et al.,J. Mol. Biol. (2007) vol. 373, p. 924-940) to show even higherexpression efficiency in E. coli was provided as Fab-SS. For the displayof this sequence in a cell-free system, a DNA fragment for bicistronicFab-PRD was artificially synthesized using codons optimized for E. coli.The synthesized fragment was incorporated into a general-purposephagemid vector pBluescript SK(+) to construct pGAv2-SS. Subsequently,the CDR 1-3 regions in the L chain and the CDR 1-3 regions in the Hchain of pGAv2-SS were replaced by the corresponding regions in pTrcFab-HH, to construct pGAv2-HH. Similarly, using pGAv2-SS and pTrcFab-TT, pGAv2-TT was constructed. Subsequently, by introducing a XhoIrecognition site into the most downstream part of the GS linkerdownstream of the L chain of pGAv2-HH without changing the amino acidsequence, pGAv2-HH xhoI was constructed. A schematic view of thestructure of the DNA fragment for bicistronic Fab-PRD is shown in FIG.13.

[Example 4] Construction of Vector for Monocistronic Fab-PRD

In pGAv2-HH xhoI and pGAv2-TT for bicistronic Fab-PRD, the region fromthe first amino acid in the ribosome stall sequence downstream of the Lchain to the first methionine in the H chain was replaced by a sequenceencoding a FLAG tag, and, via this FLAG tag, the C-terminus of the GSlinker downstream of the L chain was linked in-frame to the H chain, toconstruct vectors for monocistronic Fab-PRD, pGAv6-HH xhoI and pGAv6-TT.A schematic view of the structure of the DNA fragment for monocistronicFab-PRD is shown in FIG. 14.

[Example 5] Preparation of Template DNA

Template DNA fragments for in vitro transcription were amplified by PCRusing, as a template, each of pGAv2-HH xhoI and pGAv2-TT for bicistronicFab-PRD and each of pGAv6-HH xhoI and pGAv6-TT for monocistronicFab-PRD. As an enzyme for PCR, PrimeStarMax (Takara) was used, and, asprimers, the combination of PURE-rt-1F and PURE-3R or the combination ofSL-1F and SL-2R was used. The amplified template DNA fragments for invitro transcription were purified by phenol/chloroform extraction andisopropanol precipitation. The concentration of each template DNAfragment for in vitro transcription was calculated based on theabsorbance at 260 nm under the assumption of 1 OD=50 μg/ml. Thesequences of the template DNA fragments for in vitro transcriptionamplified from the 4 types of vectors using the primer set of SL-1F andSL-2R were as follows.

For bicistronic Fab-PRD

pGAv2-HH xhoI, SEQ ID NO:13 (for the amino acid sequence, see SEQ IDNOs:14 (L) and 15 (H))

pGAv2-TT, SEQ ID NO:16 (for the amino acid sequence, see SEQ ID NOs:17(L) and 18 (H))

For monocistronic Fab-PRD

pGAv6-HH xhoI, SEQ ID NO:19 (for the amino acid sequence, see SEQ IDNO:20)

pGAv6-TT, SEQ ID NO:21 (for the amino acid sequence, see SEQ ID NO:22)

The sequences of the primers used were as follows.

PURE-rt-1F: (SEQ ID NO: 23)(caatttcggtaatacgactcactatagggagaatttaggtgacactata gaagtg) PURE-3R:(SEQ ID NO: 24) (caggtcagacgattggccttg) SL-1F: (SEQ ID NO: 25)(caatttcggtaatacgactcactatagggagaccacaacggtttcccatttaggtgacactatagaagtg) SL-2R: (SEQ ID NO: 26)(ccgcacaccagtaaggtgtgcggcaggtcagacgattggccttgatatt cacaaacg)

[Example 6] mRNA Synthesis

mRNA synthesis was carried out using 500 ng of a template DNA fragmentand 2.7 μg of purified T7 RNA polymerase, at a scale of 50 μl. After thesynthesis reaction at 37° C. for 60 minutes, 1 μl of RNase-free DNase(Promega) was added to the reaction solution to degrade the template DNAby the reaction at 37° C. for 10 minutes. The mRNA was purified byphenol/chloroform extraction and isopropanol precipitation, anddissolved in 30 μl of nuclease-free water (Promega). The concentrationof the mRNA was calculated based on the absorbance at 260 nm under theassumption of 1 OD=40 μg/ml.

[Example 7] Translation of mRNA by Reconstruction-Type Cell-FreeTranslation System (PURE System)

The translation factors and the ribosomes constituting the PURE systemwere prepared according to the methods described in reports by Shimizuet al. (Shimizu et al., Nat Biotechnol. (2001) vol. 19, p. 751-755) andOhashi et al. (Ohashi et al., Biochem Biophys Res Commun. (2007) vol.352, p. 270-276). Translation reaction was carried out using 2 pmol ofmRNA and 20 pmol of ribosomes at a scale of 20 μl. In terms of theoxidation-reduction conditions, oxidized glutathione and reducedglutathione, and protein disulfide isomerase were removed, and 1 mM DTTwas added to perform the translation reaction under reducing conditions.After the translation reaction at 37° C. for 20 minutes, oxidizedglutathione was added at a final concentration of 2.5 mM to neutralizeDTT.

[Example 8] Biotinylation, and Confirmation of Quality of Antigen

Each of Her-2 and INFαR (R&D systems) was dissolved in PBS at 0.5 mg/ml.As a biotinylation reagent, 1.1 μl (for Her-2) or 3.6 μl (for INFαR) ofSulfo-NHS—SS-Biotin (Thermo Scientific) prepared at a concentration of0.6 mg/ml (for Her-2) or 4.0 mg/ml (for INFαR) in PBS was added to 100μl of the antigen solution, and the reaction was allowed to proceed atroom temperature of 1 hour. The reaction product was passed through agel filtration spin column to remove unreacted biotinylation reagent.Avidin (Calbiochem) was diluted to 2 μg/ml with PBS. The diluted avidinwas placed on an ELISA plate and incubated at 4° C. overnight forimmobilization on the plate. After washing the well, the biotinylatedantigen was added thereto to bind the biotinylated antigen to theavidin. After washing the well, the high-purity model Fab prepared inExample 1 was added thereto to bind the model Fab to the biotinylatedantigen. After washing the well, a 1000-fold diluted HRP-labeledanti-FLAG antibody (Sigma) as a detection antibody was added to thewell, to bind the detection antibody to the FLAG tag attached to themodel Fab. After washing the well, a colorimetric substrate for HRP wasadded thereto. When an appropriate degree of coloring was achieved, astop solution was added to the well, and the absorbance was measured atOD 405 nm. The obtained result is shown in FIG. 15. It can be seen thatthe 2 types of biotinylated antigens have simultaneous reactivity toavidin and the corresponding model Fab.

[Example 9] Selection of Fab by PRD (Fab-PRD)

By reference to the method reported by Ohashi et al. (Ohashi et al.,Biochem Biophys Res Commun. (2007) vol. 352, p. 270-276), in vitroselection of Fab was carried out by ribosome display based on the PUREsystem (PRD). Thirty microliters of M-280 streptavidin magnetic beads(DYNAL) were washed, and 5 pmol of the biotinylated antigen proteinprepared in Example 8 was added thereto, followed by allowing thereaction to proceed at room temperature for 30 minutes in order toimmobilize the bait protein on the magnetic beads. The magnetic beadswere washed to provide beads for selection. By the same procedure, beadsfor preclearing, on which the bait protein was not immobilized, wereprepared.

A small amount of Fab-HH xho was added to Fab-TT as a template DNA ormRNA to an arbitrary content, to provide a sample before selection. Thissample was used to prepare an in vitro translation product by the methoddescribed in Example 7. The prepared product was mixed with the beadsfor preclearing, and the reaction was allowed to proceed at 4° C. for 30minutes, followed by recovering the supernatant. The supernatant wasmixed with the beads for selection, and the reaction was allowed toproceed at 4° C. for 30 minutes, followed by recovering the magneticbeads. The magnetic beads were washed, and 100 μl of an elution bufferwas added thereto, followed by allowing the reaction to proceed at roomtemperature for 10 minutes. The elution buffer was prepared using (50 mMTris-Cl pH 7.6, 150 mM NaCl, 10 μg/ml budding yeast RNA) as a base, andDTT was further added thereto to a final concentration of 100 mM in thecase of reducing elution, or EDTA was added thereto to a finalconcentration of 50 mM in the case of chelate elution. The mRNA bound tothe magnetic beads via Fab was eluted by reductive cleavage of the S—Sbond in the biotin linker, in the case of reducing elution; or bydissociating the display molecule complex (complex of the 3 components,mRNA-ribosome-polypeptide) which is dependent on magnesium ions, in thecase of chelate elution. From the eluted sample, mRNA was purified byphenol/chloroform extraction and isopropanol precipitation, or usingRNaeasy RNA purification kit (QIAGEN).

By the reaction using SuperScript III (Invitrogen) as reversetranscriptase, the eluted mRNA was converted to cDNA. As a primer forthe reverse transcription reaction, PURE-2R or PURE-3R was used. Afteramplification of cDNA by PCR reaction using PrimeStarMax (Takara), theamplification product was purified by phenol/chloroform extraction andisopropanol precipitation, to provide a DNA sample after selection. AsPCR primers for amplification of the full-length sequence of mRNA, thecombination of PURE-rt-1F and PURE-3Ror the combination of SL-1F andSL-2R was used. Further, as PCR primers for amplification of a partialfragment corresponding to the region from CDR3 in the L chain to CDR1 inthe H chain, the combination of L-CDRex-1F and H-CDRex-2R was used. Thesequences of the primers used were as shown below.

(SEQ ID NO: 27) PURE-2R: (gacgattggccttgatattcacaaacg) (SEQ ID NO: 28)L-CDRex-1F: (attaaacgtaccgttgcagcaccgagc) (SEQ ID NO: 29) H-CDRex-2R:(tgagcctccaggctgaaccagaccac)

[Example 10] Confirmation of Specific Enrichment by Fab-PRD

The DNA samples before selection and after selection were digested withXhoI, and separated with 1% agarose gel, followed by staining withethidium bromide. It is expected that the DNA sample before selection ishardly digested with XhoI since it contains Fab-TT as the majorcomponent, but the DNA sample after selection has higher sensitivity toXhoI since the content of Fab-HH xho is higher. Based on such anincrease in the sensitivity of the DNA sample to XhoI, enrichment ofFab-HH DNA was confirmed. The result of Fab-HH selection by single-roundbicistronic or monocistronic Fab-PRD using Her-2 as a bait protein isshown below.

From a sample with a TT/HH ratio of 10, Fab-HH was selected bybicistronic Fab-PRD, and a partial fragment corresponding to the regionfrom CDR3 in the L chain to CDR1 in the H chain was amplified toevaluate the sensitivity to XhoI. The result is shown in FIG. 16. In thecases where a mock elution buffer (WB) which does not contain acomponent necessary for specific elution such as DTT or EDTA was used,no increase in the sensitivity to XhoI could be observed relative to theDNA sample before selection, either when the selection was carried outusing the magnetic beads untreated with biotinylated Her-2 or when theselection was carried out using the magnetic beads treated withbiotinylated Her-2. On the other hand, in the cases where the selectionwas carried out using the magnetic beads treated with biotinylated Her-2and the elution buffer containing DTT or EDTA was used, an increase inthe sensitivity to XhoI could be observed. Based on these results, itcan be seen that Fab-HH was specifically enriched.

Similarly, from DNA samples with TT/HH ratios of 10 and 100, Fab-HH wasselected by bicistronic Fab-PRD, and the selected Fab-HH was eluted withDTT, followed by amplifying the full-length sequence of mRNA in order toevaluate the sensitivity to XhoI. The result is shown in FIG. 17. In theboth cases of TT/HH ratios of 10 and 100, an increase in the sensitivityto XhoI was observed, so that it can be seen that Fab-HH wasspecifically enriched.

Further, from DNA samples with TT/HH ratios of 100 to 100,000, Fab-HHwas selected by monocistronic Fab-PRD. The result is shown in FIG. 18.In the cases of TT/HH ratios of 100 to 1000, an increase in thesensitivity to XhoI was observed, so that it can be seen that Fab-HH wasspecifically enriched.

[Example 11] Confirmation of In Vitro Translation Product ofMonocistronic Fab

By the method described in Example 7, a cell-free translation productwas prepared using mRNA for monocistronic Fab-PRD. The translationproduct was separated by SDS-PAGE with 5-20% gradient gel, andtransferred to a PVDF membrane. Western blotting was then performedusing a 1000-fold diluted HRP-labeled anti-FLAG antibody (Sigma) as adetection antibody. The result is shown in FIG. 19. It can be seen that,although the full-length translation product of scFab-TT can beobserved, the ratio of the full-length translation product is extremelysmaller than the amount of scFv-TT translated, which was used as apositive control.

[Example 12] Relationship Between Copy Number and Recovery of SecM

PCR was carried out using pGAv6-HH xhoI as a template to synthesize aDNA fragment in which the ribosome stall sequence (SecM sequence) wasduplicated into 2 copies, to provide the v6.5 fragment. By similar PCR,the v6.5-S3 fragment having 3 copies of the SecM sequence, and thev6.5-S4 fragment having 4 copies of the SecM sequence were prepared.Using each prepared template DNA fragment (100% Fab-HH), the recovery ofFab-HH by monocistronic Fab-PRD was measured. The monocistronic Fab-PRDwas carried out by the solid-phase selection described in Example 9, andthe recovered reverse transcription product was quantified byquantitative PCR using, as samples for preparing a calibration curve,the template DNA fragment at known concentrations. As detection primersto be used for the quantitative PCR, 2 types of primers, the upstreamprimers and the downstream primers, were prepared, and the recovery (thenumber of molecules of the 1st strand cDNA/the number of input RNAmolecules) was determined for each of the upstream region in thevicinity of CDR-L1 and the downstream region in the vicinity of CDR-H3.The result is shown in FIG. 20. By duplicating the SecM sequence into 2copies, the recovery of Fab-HH by Fab-PRD increased about 10-fold. Inthis experiment, increasing of the copy number of the SecM sequence to 3or more did not further increase the recovery, but it is considered thatthe recovery might further increase by increasing the copy number of theSecM sequence to 3 or more in cases where the experimental conditionsare changed by, for example, extending the period of translationreaction.

The primers used for the quantitative PCR were as follows.

Upstream primers for FabHH (SEQ ID NO: 33) RT-1F:gatattcagatgacccagagcccgagc (SEQ ID NO: 34) RT-1R: cagcttcggggcttttcctggDownstream primers for FabHH (SEQ ID NO: 35) HH-4F:ccagggaactttggttactgtttc (SEQ ID NO: 36) Model-4R:ctttaaccagacaacccagtgc

The sequences downstream of the SecM region in the v6.5, v6.5-S3 andv6.5-S4 DNA fragments used in the Fab-PRD are shown in SEQ ID NOs:37, 40and 44, respectively.

[Example 13] Relationship Between Linker Length and Enrichment Ratio

The template DNA to be used in monocistronic Fab-PRD has a total of 2linker sequences which are located between the L chain and the H chainand between the H chain and SecM. In the v6.5 fragment in Example 12,the linker between the L chain and the H chain has a length of 128 aminoacids, and the linker between the H chain and SecM has a length of 123amino acids. In order to study the effect of the linker length on the cenrichment ratio in the monocistronic Fab-PRD, the v6.5 fragment having2 copies of the SecM sequence was used as the basic structure formodification of each of Fab-HH xhoI and Fab-TT, to prepare the v7fragment by minimizing the length of the linker between the H chain andSecM, and the v8.1 fragment by minimizing the length of the linkerbetween the L chain and the H chain.

The length of the linker between the H chain and SecM in the v7 fragmentwas 20 amino acids in terms of the length from the cysteine as the lastamino acid of the H chain to the alanine as the first amino acid ofSecM. The length of the linker between the L chain and the H chain inthe v8.1 fragment was 30 amino acids in terms of the length from thecysteine at the C-terminus of the L chain to the glutamic acid at theN-terminus of the H chain. For each structure, the template DNAs weremixed at a TT/HH ratio of 100, and Fab-HH xhoI was selected by theFab-PRD by solid-phase selection described in Example 9. Using theobtained reverse transcription products as samples, the recoveries ofFab-HH and Fab-TT were measured by quantitative PCR to determine the cenrichment ratio (the recovery of Fab-HH/the recovery of Fab-TT). Asdetection primers to be used for the quantitative PCR, the downstreamprimers in the vicinity of CDR-H3 were used. The result is shown in FIG.21. Since a practical enrichment ratio was observed either in the casewhere the linker between the H chain and SecM was minimized or in thecase where the linker between the L chain and the H chain was minimized,it is considered that the linker length can be flexibly selecteddepending on the situation.

The primers used for the quantitative PCR were as follows.

Downstream primers for FabHH (SEQ ID NO: 35) HH-4F:ccagggaactttggttactgtttc (SEQ ID NO: 36) Model-4R:ctttaaccagacaacccagtgc Downstream primers for Fab-TT (SEQ ID NO: 49)TT-5F: gcaccctggttaccgtgag (SEQ ID NO: 36) Model-4R:ctttaaccagacaacccagtgc

The sequences of the linker portions in the v7 and v8.1 DNA fragmentsused in the Fab-PRD are shown in SEQ ID NOs:50 and 52, respectively.

[Example 14] Study of Minimum Required Copy Number in MonocistronicFab-PRD

The v7 fragments of Fab-HH xhoI and Fab-TT (Example 13) were prepared astemplate DNAs and used for preparation of a 10-fold dilution series ofthe Fab-TT template DNA in a carrier solution containing 0.125 mg/mlyeast RNA and 7×10⁹ molecules/μl of the Fab-HH template DNA. DNA samplescontaining 1×10⁶ molecules/μl to 1×10⁻⁵ molecules/μl of the Fab-TTtemplate DNA were subjected to amplification by single-molecule PCRusing Fab-TT-specific primers, and the PCR products were separated by5-20% gradient PAGE. It was confirmed that a Fab-TT-specific band isdetected in PCR with samples containing 1 or more Fab-TT molecules asthe template DNA while the Fab-TT-specific band is not detected in PCRwith samples containing 0.1 or less Fab-TT molecule.

From a diluted sample of the Fab-TT template DNA whose quality wasconfirmed by the single-molecule PCR, an aliquot corresponding to 400molecules was collected, and the collected sample was added to 1×10¹²molecules of the Fab-HH template DNA. RNA was synthesized from this, toprovide RNA before selection. The RNA was subjected to the Fab-PRD, andrecovery of the Fab-TT in an amount corresponding to 400 molecules wasattempted. The translation reaction was carried out at a scale of 20 μlat 30° C. for 40 minutes, and, thereafter, an equal volume of WBTBRbuffer (50 mM Tris-HCl (pH=7.6), 90 mM NaCl, 50 mM Mg(OAc)₂, 5 mg/mlBSA, 1.25 mg/ml yeast RNA, 0.5% Tween 20, 0.04 U/μl RNase Inhibitor)supplemented with 1 μM biotinylated INFαR was mixed therewith, followedby incubation at 4° C. overnight. To this mixture, an M-280 streptavidinmagnetic bead pellet in an amount corresponding to 25 μl was added, andthe Fab-TT was recovered. RNA eluted from the M-280 beads with DTT wasprovided as RNA after selection. Each of the RNAs before and afterselection by Fab-PRD was reverse transcribed, and RT-PCR was performedto amplify a Fab-TT fragment (lanes 1 and 2 in FIG. 22) withFab-TT-specific primers in the vicinity of CDR-H3, and a core fragmentcontaining important genetic information corresponding to the regionfrom CDR-L1 to CDR-H3 (lanes 4 and 5 in FIG. 22) with primers thatrecognize DNA sequences which are common between Fab-HH and Fab-TT. Theband for the core DNA fragment was recovered from 5-20% gradient gel,and reamplified by 2nd PCR (lanes 7 and 8 in FIG. 22).

Using the core DNA fragment obtained by 2nd PCR, PCR was performed usingthe Fab-TT-specific primers to amplify the Fab-TT fragment. As a result,the Fab-TT fragment was detected not only from the sample beforeselection but also from the sample after selection, so that it wasconsidered that recovery of the core fragment from the 400 molecules ofFab-TT was successful. Subsequently, for confirmation of the fact thatthe recovery was specific, the enrichment ratio of Fab-TT was measuredby quantitative PCR. The Fab-TT fragment and the core fragment wereindependently quantified, and the enrichment ratio (the relative amountof Fab-TT in the sample after selection/the relative amount of Fab-TT inthe sample before selection) was calculated. As a result, it was foundthat the content of Fab-TT was 582 times higher in the core DNA fragmentafter selection (lane 7 in FIG. 22) than in the core DNA fragment beforeselection (lane 8 in FIG. 22) (enrichment ratio=582). From theseresults, it was suggested that a target-binding clone contained in thelibrary can be specifically recovered by Fab-PRD in cases where at least400 molecules of the clone are contained. This rate of recovery isconsidered to be comparable to those of phage display systems.

The primers used were as follows.

Fab-TT-specific primers (SEQ ID NO: 54) T1P-F:gttttactattgaacgttatgcgatgggt (SEQ ID NO: 55) T1P-R:cgtagtacataccgttcgggtagttag

Primers for amplification of the core region which is common betweenFab-HH and Fab-TT

Core-1F: gcgcaagcgttggtgatc (SEQ ID NO: 56) Core-1R:gctcggacctttggtgcttg (SEQ ID NO: 57)

[Example 15] Construction of Ymacs-Primary Library (First Half of FirstStep of Affinity Maturation)

As an example of application the Fab-PRD, affinity maturation of Fab-TT,which is one of the model Fabs, was attempted. On the antigen-antibodybinding interface, there are not less than 100 van der Waals forces, andseveral hydrogen bonds and salt bridges. In order to realize overalloptimization of the network of these interactions, a fundamental 2-stepstrategy called Ymacs was employed, wherein mutations in CDRs which areconsidered to be beneficial for increasing the affinity are searched inthe first step, and the optimal combination of these mutations issearched in the second step.

As an operation of the first half of the first step, a library(Ymacs-primary library) for matrix scanning of the CDR positionscorresponding to 50 amino acids was constructed such that every singleamino acid among the total of 50 amino acids constituting the CDRs ofFab-TT was randomized into 20 types of amino acids.

First, in order to substitute each of the total of 50 amino acidsconstituting the CDRs of Fab-TT with the NNK codon, a total of 50 typesof forward primers for introduction of single amino acid substitutionswere synthesized. These forward primers for introduction of single aminoacid substitutions were designed such that a 15-bp annealing region isplaced upstream of the NNK codon, and a 12-bp annealing region is placeddownstream of the NNK codon. As examples, the primers corresponding tothe most upstream site and the most downstream site among the 50 siteswhere the single amino acid substitutions were introduced are shownbelow.

FabTT Ymacs-1 L1-1: tgtcgtgcaagccagNNKattaaaaattat (SEQ ID NO:58) (theprimer for introduction of a single amino acid substitution at the firstamino acid in CDR1 in the L chain)

FabTT Ymacs-1 H3-12: atgtactacgttatgNNKtattggggtcag (SEQ ID NO:59) (theprimer for introduction of a single amino acid substitution at the 12thamino acid in CDR3 in the H chain)

By performing PCR using pGAv6.5-Fab-TT as a template and each of theabove-described 50 types of forward primers for introduction of singleamino acid substitutions in combination with the PURE-3R primer, 50types of mutated downstream DNA fragments were individually synthesized.The PCR products were separated with 1% agarose gel, and stained withethidium bromide to confirm their bands. Subsequently, a commonconstant-sequence upstream DNA fragment was synthesized by PCR, for useas a template in the reaction to extend the reverse strand of themutated downstream DNA fragment in the upstream direction to provide afull-length DNA. The constant-sequence upstream DNA fragment wassynthesized by PCR using PURE-rt-1F and H3checkR Not TA6 as primers suchthat the fragment has a misanneal region in the 3′-end side in order toavoid production of the full-length DNA from its own forward strand. ThePCR product was separated with 1% agarose gel, and stained with ethidiumbromide for confirmation of its band.

PURE-rt-1F: (SEQ ID NO: 60)caatttcggtaatacgactcactatagggagaatttaggtgacactatag aagtgH3checkR Not TA6: (SEQ ID NO: 61)tatatatatatagcggccgcagaactgccggaaaggtatg

A summary of synthesis of the single-position/single-amino acidsubstitution library is shown in FIG. 23. Two cases of the synthesiswherein the site into which the amino acid substitution was introducedis located most upstream and most downstream are shown. After mixing theconstant-sequence upstream DNA fragment (A and B in the figure) and themutated downstream DNA fragment (C and D in the figure) together,asymmetric PCR was carried out in the presence of the PURE-rt-1F primer(E in the figure). By this, the C strand as the reverse strand of themutated downstream DNA fragment is extended in the upstream direction toproduce a full-length DNA, and the full-length C strand is convertedinto a double strand by PURE-rt-1F. By mixing 4 μl of each mutateddownstream DNA fragment with 0.6 μl of the constant-sequence upstreamDNA fragment and performing asymmetric PCR at a scale of 50 μl, 50 typesof single-position/single-amino acid substitution libraries weresynthesized. The obtained PCR product was separated with 1% agarose gel,and stained with ethidium bromide for confirmation of its band.

The single-position/single-amino acid substitution libraries weredivided into the L chain group and the H chain group, and equal volumesof the libraries in each group were mixed together, to provide theL-chain Ymacs-primary library and the H-chain Ymacs-primary library. Asshown in FIG. 24, the L-chain Ymacs-primary library is constituted by 19types of, and the H-chain Ymacs-primary library is constituted by 31types of, single-position/single-amino acid substitution libraries.

[Example 16] Selection and Next-Generation Sequence Analysis ofYmacs-Primary Libraries (Latter Half of First Step of AffinityMaturation)

As an operation of the latter half of the first step, the Ymacs-primarylibraries of the L chain and the H chain were independently selected byFab-PRD, and the result of next-generation sequence analysis of theobtained DNA samples was used to identify mutations in CDRs which areconsidered to be beneficial for affinity improvement.

In the selection of the Ymacs-primary libraries by Fab-PRD, thesolid-phase selection described in Example 9 and the liquid-phaseselection described in Example 14 were used in combination. In round 1,solid-phase selection was carried out, and, in round 2, solid-phaseselection was carried out followed by a long period (13 h) of washing.In the final round, round 3, liquid-phase selection was carried out at abait concentration of 1 nM. In every round, 1×10¹² molecules of RNA weretranslated at a scale of 20 μl. In the reverse transcription-PCR, thecore DNA fragment described in Example 14 was recovered. To the core DNAfragment, an appropriate upstream fragment and downstream fragment wereadded, and overlapping extension-PCR was performed using PURE-rt-1F andPURE-3R as primers to synthesize the full-length DNA fragment, which wasthen purified and used as the template DNA in the following round.

The nucleotide sequences of the CDR 1-3 regions in each of the L chainand the H chain in the core DNA fragment obtained in round 3 wereanalyzed with a Roche GS FLX next-generation sequencer, and 1812 readsof the L-chain CDR 1-3 sequences in the sample derived from the L-chainlibrary and 2288 reads of the H-chain CDR 1-3 sequences in the samplederived from the H-chain library were collected as nucleotide sequencedata. From these data, 468 reads from the L chain and 1293 reads fromthe H chain having a continuous ORF in the CDR1-3 regions were selectedas effective reads, and mutations at the CDR positions for a total of 50amino acids, that is, 19 amino acids in the L chain and 31 amino acidsin the H chain constituting the CDRs, were summarized. The number oftimes each mutation was counted was summarized in the matrix table shownin FIG. 25, wherein the 20 types of amino acids are arranged in thelongitudinal direction and the total of 50 CDR positions are arranged inthe horizontal direction. Mutations with a large number of counts,except for the parent amino acid at each CDR position, were determinedto be potentially beneficial mutations.

[Example 17] Construction of Ymacs-Secondary Library (First Half ofSecond Step of Affinity Maturation)

As an operation of the first half of the second step, a Ymacs-secondarylibrary was constructed by combining the beneficial mutations identifiedin Example 16. As the beneficial mutations, a total of 21 mutations at atotal of 12 CDR positions were employed, and mixed-base codons weredetermined such that each of these can encode the parent amino acid andthe beneficial mutant amino acid(s) at the respective CDR positions.Some of the mixed-base codons may introduce an unintended amino acidother than the parent amino acid and the beneficial mutant amino acids.The employed beneficial mutations and the mixed-base codons aresummarized in FIG. 26. The theoretical diversity of the Ymacs-secondarylibrary was 1.9×10⁷ based on the nucleic acid-level estimation, and4.9×10⁶ based on the protein-level estimation. Since the 12 CDRpositions were dispersed among all 6 CDRs, one mutation-introducingforward primer for each CDR, that is, a total of 6 mutation-introducingforward primers, were synthesized. Using these, a total of 6 mutated DNAfragments, that is, the fragment 2 to fragment 7 shown in FIG. 27, weresynthesized. A total of 7 mutated DNA fragments which include these 6fragments and the fragment 1 were linked together by overlappingextension reaction to prepare a full-length DNA, which was thenamplified by PCR reaction. After purification of the obtained PCRproduct, occurrence of randomization as designed with the mixed bases atthe sites of introduction of mutations was confirmed, to provide aYmacs-secondary library.

The following are the mutation-introducing forward primers.

FabTT Ymacs-2 L1-Fwd: (SEQ ID NO: 62)tgtcgtgcaagccaggatattaaaaattatttgWCTtggtatcaacaaca a (L-chain CDR1)FabTT Ymacs-2 L2-Fwd: (SEQ ID NO: 63)gccccgaagccactgatttatGSTggttctaaccgccaatctggagttcc t (L-chain CDR2)FabTT Ymacs-2 L3-Fwd: (SEQ ID NO: 64)acctattattgccaacaaactKMTRNMtaccctatcacctttggccag (L-chain CDR3)FabTT Ymacs-2 H1-Fwd: (SEQ ID NO: 65)agctgtgcagcaagcggttttASAattGRGcgttatgcgatgRSTtgggt gcgtcaggct(H-chain CDR1) FabTT Ymacs-2 H2-Fwd: (SEQ ID NO: 66)ggcctggaatgggttggtacgatttatcctKDSRSCgattatRBYgatta tgccgatagc(H-chain CDR2) FabTT Ymacs-2 H3-Fwd: (SEQ ID NO: 67)tactactgcgctcgctctaactacccgaacggtMTGKRCtacgttatgga atat (H-chain CDR3)

[Example 18] Selection of Ymacs-Secondary Library and Analysis ofAffinity of Clones (Latter Half of Second Step of Affinity Maturation)

As an operation of the latter half of the second step, theYmacs-secondary library was selected by Fab-PRD, and the obtained DNAswere cloned into a secretory expression vector for E. coli.High-affinity clones were selected by ELISA and SPR (ProteOn XPR36,BioRad), and 2 representative clones of Fab-TT mutants (Ymacs #10 and#19 in FIGS. 29 to 32) were subjected to measurement of KD using KinExA(KinExA 3200, Sapidyne).

Selection of the Ymacs-secondary library by Fab-PRD was carried out by atotal of 5 rounds of selection, in each of which liquid-phase selectionwas performed. The operation proceeded from round 1 to 5 while the baitconcentration was gradually decreased from 100 nM to 60 nM, 20 nM, 1 nM,100 pM and then 10 pM, to increase the selection pressure. The core DNAfragment recovered in round 5 was cloned such that the core region ofFab-HH in a monocistronic scFab-HH expression vector was replaced by thefragment (FIG. 28). Using the culture supernatants of 48 clones randomlypicked up, ELISA was carried out with INFαR as an antigen. Afterconfirming that the hit rate in ELISA was about 50%, the remainingseveral hundred colonies were recovered at once in the polyclonal state,and their plasmids were purified. The GS linker region between the Lchain and the H chain of the scFab-TT mutant encoded by each plasmid wasreplaced by an untranslated sequence-secretion signal fragment forbicistronic expression, to convert the expression mode from themonocistronic type to the bicistronic type (FIG. 28). By this operation,a vector that expresses the Fab-TT mutant as a naturally-occurring Fabrather than scFab was constructed, and cloning was carried out again.Using the culture supernatants of 96 clones randomly picked up, ELISAwas carried out with INFαR as an antigen. The ELISA hit clones weresubjected to determination of the sequences of their CDRs (FIG. 29). TheELISA hit clones were subjected to screening by SPR with a binding timeof 1 minute and a dissociation time of 30 minutes, to selecthigh-affinity clones using Koff as an index (FIG. 30). As a result of anattempt to measure the affinities of the parent Fab Fab-TT and anaffinity-improved mutant Ymacs #10 by SPR, KD was determined as follows:Fag-TT, KD=7.28×10⁻⁹; Ymacs #10, KD<1.57×10⁻¹¹. Thus, an about 460-foldincrease in the affinity was observed (FIG. 31). Subsequently, as aresult of an attempt to measure the affinities of 2 clones, Ymacs#10 andYmacs #19, having highly improved affinities by KinExA, KD wasdetermined as follows: Ymacs #10, KD=1.87×10⁻¹¹; Ymacs #19,KD=3.45×10⁻¹². Thus an about 2100-fold increase in the affinity wasobserved for Ymacs #19 (FIG. 32).

The experiment for measurement of KD of Ymacs #19 by KinExA was carriedout at 2 different Fab concentrations, 35 pM and 350 pM. In tams of theconcentration of INFαR as an antigen, a 2-fold dilution series from 2 nMto 976 fM was prepared for 35 pM Fab, and a 2-fold dilution series from5 nM to 2.44 pM was prepared for 350 pM Fab. Each sample was incubatedat room temperature for 48 hours until the antigen-antibody reactionreaches equilibrium, and the fraction of free Fab after reaching theequilibrium was quantified by KinExA using azlactone beads on whichINFαR was immobilized. The fraction of free Fab (ordinate) was plottedagainst the antigen concentration (abscissa), to draw 2 dose-responsecurves corresponding to the different concentrations of Fab. The curveswere subjected to global fitting analysis by KinExA Pro Software, tocalculate KD.

[Example 19] Selection of Fabs by CIS Display (Fab-CIS)

By reference to a report by Odegrip et al. (Odegrip et al., Proc NatlAcad Sci USA. (2004) vol. 101, p. 2806-2810), selection of Fabs by CISdisplay was attempted. As shown in FIG. 8, overlapping extension-PCR wasperformed to add a promoter sequence for E. coli RNA polymerase and theRepA-CIS-ori sequence in the upstream region and the downstream region,respectively, of the scFab-coding region in the v7 fragment, to providethe v10.1 fragment. Since the RepA-coding sequence was different betweenthe sequence reported by Odegrip et al. and the sequence of GenBankV00351, the sequence of V00351 was employed. A mixture wherein v10.1fragments of Fab-HH xhoI and Fab-TT were mixed at a TT/HH ratio of 10was used as a template DNA for in vitro transcription/translationreaction. Purified template DNA (1.2 μg) was subjected to an E.coli-derived S30-based transcription/translation system (25 μL, derivedfrom the E. coli SL119 strain, for linear DNA, L1030, Promega), tosynthesize the display molecule complex at 30° C. for 40 minutes.Thereafter, by reference to the report by Odegrip et al., Fab-HH wasrecovered by liquid-phase selection in the same manner as in Example 14,and PCR was carried out by using the eluted DNA as a template and Cis-1Fand Cis-6R as primers, to amplify the full-length fragment. By the sameevaluation system as in Example 10 using the sensitivity to XhoI as anindex, the performance of the Fab-HH selection system by CIS display waschecked. The result is shown in FIG. 33. When the selection was carriedout without addition of biotinylated Her-2, no increase in thesensitivity to XhoI was observed relative to the DNA sample beforeselection. On the other hand, when the selection was carried out byaddition of biotinylated Her-2, an increase in the sensitivity to XhoIcould be observed. From these results, it can be seen that Fab-HH wasspecifically enriched.

Further, a mixture wherein v10.1 fragments of Fab-HH xhoI and Fab-TTwere mixed at a TT/HH ratio of 100 was used to perform the sameexperiment, to study the optimal transcription/translation time. Theresult is shown in FIG. 34. With every transcription/translation timestudied, specific enrichment of Fab-HH could be observed. With atranscription/translation time of 10 minutes, the enrichment ratio wasinsufficient. However, the enrichment ratio sufficiently increased with20 minutes to 40 minutes of the transcription/translation, and the ratiotended to gradually decrease after 80 minutes to 120 minutes of thetranscription/translation. From these results, the optimaltranscription/translation time was considered to be 20 minutes to 40minutes.

The sequences of the primers for amplification of the full-length v10.1fragment were as follows.

Cis-1F (SEQ ID NO: 68) cagttgatcggcgcgagatttaatcgccgc Cis-6R(SEQ ID NO: 69) cgtaagccggtactgattgatagatttcaccttacccatc

INDUSTRIAL APPLICABILITY

The present invention is useful in the fields of genetic engineering,protein engineering and the like. Fabs obtained by the method of thepresent invention are useful in the fields of diagnosis, medical care,research and the like.

1. A method for maximizing the affinity of a target-substance-bindingprotein to a target substance, said method comprising the steps of: (I)constructing single-position libraries wherein one amino acid among allamino acid positions constituting a target-substance-binding site in atarget-substance-binding protein is randomized to all the 20 types ofnaturally-occurring amino acids, to provide as many single-positionlibraries as the number of the all amino acid positions; (II)constructing a primary library by integrating all of, or an appropriateunit of, these single-position libraries; (III) selecting said primarylibrary using a protein display system based on the affinity to atarget; (IV) determining the polynucleotide sequence information of saidselected sample of the primary library; (V) extracting single amino acidsubstitutions frequently observed in said nucleotide sequenceinformation; (VI) constructing a secondary library comprisingcombinations of said frequently observed single amino acidsubstitutions; and (VII) selecting said secondary library using aprotein display system based on the affinity to a target.
 2. The methodaccording to claim 1, wherein the step of determining the polynucleotidesequence information of said c selected sample of the primary library iscarried out using a next-generation sequencer.
 3. The method accordingto claim 2, wherein said target-substance-binding protein is afull-length antibody or an antibody fragment and saidtarget-substance-binding site is a CDR region.
 4. The method accordingto claim 3, wherein said protein display system is ribosome display, CISdisplay, mRNA display, phage display, bacterial surface display, yeastcell surface display, or cell surface display with a higher eukaryote.5. The method according to claim 2, wherein said protein display systemis ribosome display, CIS display, mRNA display, phage display, bacterialsurface display, yeast cell surface display, or cell surface displaywith a higher eukaryote.
 6. The method according to claim 1, whereinsaid target-substance-binding protein is a full-length antibody or anantibody fragment and said target-substance-binding site is a CDRregion.
 7. The method according to claim 6, wherein said protein displaysystem is ribosome display, CIS display, mRNA display, phage display,bacterial surface display, yeast cell surface display, or cell surfacedisplay with a higher eukaryote.
 8. The method according to claim 1,wherein said protein display system is ribosome display, CIS display,mRNA display, phage display, bacterial surface display, yeast cellsurface display, or cell surface display with a higher eukaryote.